U.S. patent number 6,645,492 [Application Number 09/848,585] was granted by the patent office on 2003-11-11 for methods of treating asthma with interleukin-9 receptor antibodies.
This patent grant is currently assigned to Genaera Corporation. Invention is credited to U. Prasad Kari, Roy Clifford Levitt, W. Lee Maloy, Nicholas C. Nicolaides.
United States Patent |
6,645,492 |
Levitt , et al. |
November 11, 2003 |
Methods of treating asthma with interleukin-9 receptor
antibodies
Abstract
A C to T DNA variation at position 3365 in exon 5 of the human
Asthma Associated Factor 1 (AAF1) produces the predicted amino acid
substitution of a methionine for a threonine at codon 117 of AAF1.
When this substitution occurs in both alleles in one individual, it
is associated with less evidence of atopic allergy including
asthma, fewer abnormal skin test responses, and a lower serum total
IgE. Thus, applicant has identified the existence of a
non-asthmatic, non-atopic phenotype characterized by methionine at
codon 117 when it occurs in both AAF1 gene products in one
individual.
Inventors: |
Levitt; Roy Clifford (Ambler,
PA), Maloy; W. Lee (Lansdale, PA), Kari; U. Prasad
(Hatfield, PA), Nicolaides; Nicholas C. (Media, PA) |
Assignee: |
Genaera Corporation (Plymouth
Meeting, PA)
|
Family
ID: |
21702397 |
Appl.
No.: |
09/848,585 |
Filed: |
May 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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325571 |
Jun 4, 1999 |
6261559 |
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874503 |
Jun 13, 1997 |
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697419 |
Aug 23, 1996 |
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Current U.S.
Class: |
424/139.1;
424/141.1; 424/143.1; 424/144.1; 514/826; 514/885; D24/110 |
Current CPC
Class: |
A61P
11/00 (20180101); C07K 14/5425 (20130101); A61K
31/145 (20130101); A61P 11/06 (20180101); A61K
31/13 (20130101); A61K 31/00 (20130101); A61P
37/08 (20180101); C07K 14/7155 (20130101); A61K
38/00 (20130101); Y10S 514/885 (20130101); Y10S
514/826 (20130101) |
Current International
Class: |
A61K
31/13 (20060101); A61K 31/145 (20060101); C07K
14/715 (20060101); A61K 31/00 (20060101); C07K
14/435 (20060101); C07K 14/54 (20060101); A61K
38/00 (20060101); C07K 016/28 () |
Field of
Search: |
;514/2,8,12,885,826
;424/85.1,85.2,139.1,141.1,143.1,144.1 ;D24/110 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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A2-0361284 |
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Apr 1990 |
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EP |
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WO90/14432 |
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Nov 1990 |
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WO |
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WO91/10738 |
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Jul 1991 |
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WO |
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WO91/14767 |
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Oct 1991 |
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WO |
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WO 92/05698 |
|
Apr 1992 |
|
WO |
|
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|
Primary Examiner: Mertz; Prema
Attorney, Agent or Firm: Morgan, Lewis & Bockius LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This is a continuation application of U.S. application 09/325,571,
now U.S. Pat. No. 6,261,559, which is a continuation of U.S.
application Ser. No. 08/874,503 now abandoned, which claims the
benefit of U.S. provisional application 60/002,765.
Claims
What is claimed is:
1. A method of treating a patient suffering from asthmatic symptoms
comprising administering an amount of an antibody specific for
human interleukin-9 receptor effective to reduce the asthmatic
symptoms.
2. A method of claim 1 wherein the antibody binds to the
interleukin-9 receptor and inhibits binding of interleukin-9 to the
interleukin-9 receptor.
3. A method of claim 1 wherein the antibody down-regulates the
activity of interleukin-9 in the patient.
4. A method of claim 1 wherein the antibody reduces bronchial
hyperresponsiveness in the patient.
5. A method of claim 1 wherein the antibody reduces eosinophilia in
the lungs of the patient.
6. A method according to claim 1 wherein the antibody is
administered by one or more of the routes selected from the group
consisting of intravenous, intraperitoneal, inhalation,
intramuscular, subcutaneous and oral.
7. A method of any one of claims 1-6 wherein the antibody is a
monoclonal antibody.
8. A method of any one of claims 1-6 wherein the antibody is
administered by inhalation.
9. A method of claim 7 wherein the antibody is administered by
inhalation.
10. A method of treating asthma in a patient comprising
administering by inhalation an effective amount of an antibody that
specifically binds to the human interleukin-9 receptor.
11. A method of claim 10 wherein the antibody is a monoclonal
antibody.
12. An inhalation device that delivers to a patient a
therapeutically effective amount of an antibody that specifically
binds to the human interleukin-9 receptor.
13. An inhalation device of claim 12 wherein the antibody inhibits
binding of interleukin-9 to the interleukin-9 receptor.
14. An inhalation device of claim 12 wherein the antibody
down-regulates the activity of interleukin-9 in the patient.
15. An inhalation device of claim 12 wherein the antibody reduces
bronchial hyperresponsiveness in the patient.
16. An inhalation device of claim 12 wherein the antibody reduces
eosinophilia in the lungs of the patient.
17. An inhalation device of any one of claims 12-16 wherein the
antibody is a monoclonal antibody.
Description
FIELD OF THE INVENTION
This invention relates to regulating IL-9 activity and treating
atopic allergies and related disorders like asthma, based upon the
relationship between IL-9 and its receptor.
BACKGROUND OF THE INVENTION
Inflammation is a complex process in which the body's defense
system combats foreign entities. While the battle against foreign
entities may be necessary for the body's survival, some defense
systems improperly respond to foreign entities, even innocuous
ones, as dangerous and thereby damage surrounding tissue in the
ensuing battle.
Atopic allergy is an ecogenetic disorder, where genetic background
dictates the response to environmental stimuli. The disorder is
generally characterized by an increased ability of lymphocytes to
produce IgE antibodies in response to ubiquitous antigens.
Activation of the immune system by these antigens leads to allergic
inflammation and may occur after ingestion, penetration through the
skin, or after inhalation. When this immune activation occurs and
pulmonary inflammation ensues this disorder is broadly
characterized as asthma. Certain cells are critical to this
inflammatory reaction and they include T cells and antigen
presenting cells, B cells that produce IgE, and mast
cells/basophils and eosinophils that bind IgE. These inflammatory
cells accumulate at the site of allergic inflammation and the toxic
products they release contribute to the tissue destruction related
to the disorder.
While asthma is generally defined as an inflammatory disorder of
the airways, clinical symptoms arise from intermittent air flow
obstruction. It is a chronic disabling disorder that appears to be
increasing in prevalence and severity.sup.1. It is estimated that
30-40% of the population suffer with atopic allergy, and 15% of
children and 5% of adults in the population suffer from
asthma..sup.1 Thus, an enormous burden is placed on our health care
resources.
The mechanism of susceptibility to atopy and asthma remains
unknown. Interestingly, while most individuals experience similar
environmental exposures, only certain individuals develop atopic
allergy and asthma. This hypersensitivity to environmental
allergens known as "atopy" is often indicated by elevated serum IgE
levels or abnormally great skin test response to allergens in
atopic individuals as compared to nonatopics..sup.10 Strong
evidence for a close relationship between atopic allergy and asthma
is derived from the fact that most asthmatics have clinical and
serologic evidence of atopy..sup.4-9 In particular, younger
asthmatics have a high incidence of atopy..sup.10 In addition,
immunologic factors associated with an increase in serum total IgE
levels are very closely related to impaired pulmonary
function..sup.3
Both the diagnosis and treatment of these disorders are
problematic..sup.1 The assessment of inflamed lung tissue is often
difficult, and frequently the source of the inflammation cannot be
determined. Without knowledge of the source of the airway
inflammation and protection from the inciting foreign environmental
agent or agents, the inflammatory process cannot be interrupted. It
is now generally accepted that failure to control the pulmonary
inflammation leads to significant loss of lung function over
time.
Current treatments suffer their own set of disadvantages. The main
therapeutic agents, .beta. agonists, reduce the symptoms, i.e.,
transiently improve pulmonary functions, but do not affect the
underlying inflammation so that lung tissue remains in jeopardy. In
addition, constant use of .beta. agonists results in
desensitization which reduces their efficacy and safety..sup.2 The
agents that can diminish the underlying inflammation, the
anti-inflammatory steroids, have their own known list of
disadvantages that range from immunosuppression to bone
loss..sup.2
Because of the problems associated with conventional therapies,
alternative treatment strategies have been evaluated..sup.65-66
Glycophorin A,.sup.64 cyclosporin,.sup.65 and a nonapeptide
fragment of IL-2,.sup.63 all inhibit interleukin-2 dependent T
lymphocyte proliferation and therefore, IL-9 production,.sup.51
however, they are known to have many other effects..sup.2 For
example, cyclosporin is used as a immunosuppressant after organ
transplantation. While these agents may represent alternatives to
steroids in the treatment of asthmatics,.sup.63-66 they inhibit
interleukin-2 dependent T lymphocyte proliferation and potentially
critical immune functions associated with homeostasis. What is
needed in the art is the identification of a pathway critical to
the development of asthma that explains the episodic nature of the
disorder and the close association with allergy that is downstream
of these critical immune functions. Nature demonstrated that this
pathway is the appropriate target for therapy since biologic
variability normally exists at this pathway and these individuals
are otherwise generally not immunocompromised or ill except for
their symptoms of atopy.
Because of the difficulties related to the diagnosis and treatment
of asthma, the complex pathophysiology of this disorder is under
intensive study. Although this disorder is heterogeneous and may be
difficult to define because it can take many forms, certain
features are found in common among asthmatics. Examples of such
traits include elevated serum IgE levels, abnormal skin test
response to allergen challenge, bronchial hyperresponsiveness
(BHR), bronchodilator reversibility, and airflow
obstruction..sup.3-10 These expressions of these asthma related
phenotypes may be studied as quantitative or qualitative
measures.
Elevated IgE levels are also closely correlated with BHR, a
heightened bronchoconstrictor response to a variety of
stimuli..sup.4, 6, 8, 9 BHR is believed to reflect the presence of
airway inflammation,.sup.6, 8 and is considered a risk factor for
asthma..sup.11-12 BHR is accompanied by bronchial inflammation and
an allergic diathesis in asthmatic individuals..sup.13-21 Even in
children with no symptoms of atopy and asthma, BHR is strongly
associated with elevated IgE levels..sup.19
A number of studies document a heritable component to atopy and
asthma..sup.4, 10, 21 However, family studies have been difficult
to interpret since these disorders are significantly influenced by
age and gender, as well as many environmental factors such as
allergens, viral infections, and pollutants..sup.22-24 Moreover,
because there is no known biochemical defect associated with
susceptibility to these disorders, the mutant genes and their
abnormal gene products can only be recognized by the anomalous
phenotypes they produce. Thus, an important first step in isolating
and characterizing a heritable component is identifying the
chromosomal locations of the genes.
Cookson et al. provided the first description of a genetic
localization for inherited atopy..sup.25 These investigators
described evidence for genetic linkage between atopy and a single
marker on a specific chromosomal region designated 11q13.1. Later,
they suggested evidence of maternal inheritance for atopy at this
locus..sup.26 Although maternal inheritance genetic imprinting had
been observed for atopy, it had never been explained previously.
However, efforts to confirm this linkage have not been generally
successful..sup.27-31
Recently, the .beta. subunit of the high-affinity IgE receptor was
mapped to chromosome 11q, and a putative mutation associated with
atopy has been described in this gene..sup.32, 33 However, because
of the difficulties by others of replicating this linkage, the
significance of this gene and polymorphism remains unclear. While
additional studies will be required to confirm whether this
putative mutation causes atopy in the general population, data
collected so far suggests this polymorphism is unlikely to
represent a frequent cause of atopy.
Because serum IgE levels are so closely associated with the onset
and severity of allergy and asthma as clinical disorders, attention
has focused on studies of the genetic regulation of serum total IgE
levels. While past studies have provided evidence for Mendelian
inheritance for serum total IgE levels,.sup.34-38 an indication of
the existence of one regulatory gene, others have found evidence
for polygenic inheritance of IgE, i.e., existence of several
responsible genes..sup.39
Artisans have found several genes that may be important in the
regulation of IgE and the development or progression of bronchial
inflammation associated with asthma on chromosome 5q. They include
genes encoding several interleukins, such as IL-3, IL-4, IL-5,
IL-9, IL-13, granulocyte macrophage colony stimulating factor GM
CSF, a receptor for macrophage colony stimulating factor CSF-1R,
fibroblast growth factor acidic FGFA, as well as others..sup.40
Recent evidence from family studies suggests genetic linkage
between serum IgE levels and DNA markers in the region of these
candidate genes on chromosome 5q..sup.41, 42 Together, these
investigations suggest that one or more major genes in the vicinity
of the interleukin complex on chromosome 5q regulates a significant
amount of the observed biologic variability in serum IgE that is
likely to be important in the development of atopy and asthma.
Linkage (sib-pair analyses) was also used previously to identify a
genetic localization for BHR..sup.79 Because BHR was known to be
associated with a major gene for atopy, chromosomal regions
reported to be important in the regulation of serum IgE levels were
examined..sup.42 Candidate regions for atopy have been identified
by linkage analyses. These studies identified the existence of a
major gene for atopy on human chromosome 5q31-q33..sup.42
Therefore, to determine the chromosomal location of a gene(s)
providing susceptibility to BHR, which would be coinherited with a
major gene for atopy, experiments were carried out using linkage
analyses between BHR and genetic markers on chromosome 5q..sup.42,
79, 82 Individuals with BHR were identified by responsiveness to
histamine. Markers useful for mapping asthma-related genes are
shown in FIG. 1.
Specifically, gene candidates for asthma, bronchial
hyperresponsiveness, and atopy are shown (right) in their
approximate location relative to the markers shown. The map
includes the interleukin genes IL-4, IL-13, IL-5, and IL-3; CDC25,
cell division cycle-25; CSF2, granulocyte-macrophage colony
stimulating factor (GMCSF); EGR1 early growth response gene-1;
CD14, cell antigen 14; ADRB2, the .beta.2-adrenergic receptor;
GRL1, lymphocyte-specific glucocorticoid receptor; PDGFR,
platelet-derived growth factor receptor. Bands 5q31-q33 extend
approximately from IL-4 to D5S410. The distances reported are
sex-averaged recombination fractions.
Affected sib-pair analyses demonstrated statistically significant
evidence for linkage between BHR and D5S436, D5S658, and several
other markers located nearby on chromosome 5q31-q33..sup.79 These
data strongly supported the hypothesis that one or more closely
spaced gene(s) on chromosome 5q31-q33 determine susceptibility to
BHR, atopy, and asthma..sup.79, 80, 81, 82
Recently linkage has also been demonstrated between the asthma
phenotype and genetic markers on chromosome 5q31-q33..sup.83 This
region of the human genome was evaluated for linkage with asthma
because of the large number of genes representing reasonable
positional candidates for providing genetic susceptibility for
atopy and BHR.
Linkage was demonstrated using the methods described above..sup.42,
83 Specifically, 84 families were analyzed from the Netherlands
with both sib-pair and LODs for markers from this same region of
chromosome 5q previously shown to be linked to BHR and
atopy..sup.42, 83 An algorithm was used to categorize obstructive
airways disease in the asthmatic probands and their families. This
classification scheme was based, as described previously, on the
presence or absence of BHR to histamine, respiratory symptoms,
significant smoking history (>5 pack years), atopy as defined by
skin test response, airway obstruction (FEV1% predicted<95% CI)
and reversibility to a bronchodilator (>9% predicted).
Evidence was found for linkage between asthma and markers on
chromosome 5q by affected sib pair analysis (N=10, P<0.05) and
by maximum likelihood analysis with a dominant model for the asthma
phenotype.sup.83.
Asthma was linked to D5S658 with a maximal LOD of 3.64 at
.theta.=0.03, using a dominant model (class 1 affected, class 2-4
uncertain, class 5 unaffected) with a gene frequency of 0.015
(prevalence of 3%). A maximal LOD of 2.71 at .theta.=0.0 was
observed for D5S470 which is approximately 5 cM telomeric, or away
from IL-9, relative to D5S436..sup.83
Subsequent to the original filing of this application, IL-9 or a
gene nearby was suggested as likely to be important use atopy and
asthma..sup.43 The IL-9 suggestion was based on a strong
correlation in a randomly ascertained population between log serum
total IgE levels and alleles of a genetic marker in the IL-9
gene..sup.43 This type of association with one or more specific
alleles of a marker is termed "linkage disequilibrium", and
generally suggests that a nearby gene determines the biologic
variability under study..sup.44
The IL-9 gene has been mapped to the q31-q33 region of chromosome
5..sup.40 Only a single copy of the gene is found in the human
genome..sup.45, 46 Structural similarity has been observed for the
human and murine IL-9 genes..sup.45, 46 Each gene consists of five
exons and four introns extending across approximately four Kb of
DNA. Expression of the gene appears to be restricted to activated T
cells..sup.45, 46
The functions of IL-9 now extend well beyond those originally
recognized. While IL-9 serves as a T cell growth factor, this
cytokine is also known to mediate the growth of erythroid
progenitors, B cells, mast cells, and fetal thymocytes..sup.45, 46
IL-9 acts synergistically with IL-3 in causing mast cell activation
and proliferation..sup.47 This cytokine also potentiates the IL-4
induced production of IgE, IgG, and IgM by normal human B
lymphocytes..sup.48 IL-9 also potentiates the IL-4 induced release
of IgE and IgG1 by murine B lymphocytes..sup.49 A critical role for
IL-9 in the mucosal inflammatory response to parasitic infection
has also been demonstrated..sup.50, 51
In addition to IL-9, chromosome 5q bears numerous other gene
candidates including IL-3, IRF1, EGR1, ITK, GRL1, ADRB2, CSF1R,
FGFA, ITGA2, CD14, PDGFR, CDC25, CSF2, IL-4, IL-5, IL-12B, and
IL-13. These may all be important in atopic allergy and as
potential targets for therapeutic development. Moreover, the art
lacks any knowledge regarding how the sequence of IL-9 or the
function of IL-9 specifically correlates with atopic allergy,
asthma, or bronchial hyperresponsiveness. Without such knowledge,
artisans would not know how or whether to use IL-9 to either
diagnose or treat these disorders.
The art does provide that IL-9 is a novel cytokine having an
apparent molecular weight of approximately between 20 to 30 kD as
determined by sodium dodecyl sulfate polyacrylamide gel
electrophoresis under reducing conditions. It is produced as a 144
amino acid protein, that is processed to a 126 amino acid
glycoprotein. Yang et al..sup.85 disclose that the DNA sequence
encoding IL-9 comprises approximately 630 nucleotides, with
approximately 450 nucleotides in the proper reading frame for the
protein.
It is also known in the art that multiple protein isoforms may be
generated from a single genetic locus by alternative splicing.
Alternative splicing is an efficient mechanism by which multiple
protein isoforms may be generated from a single genetic locus.
Alternative splicing is used in terminally differentiated cells to
reversibly modify protein expression without changing the genetic
content of the cells. These protein isoforms are preferentially
expressed in different tissues or during different states of cell
differentiation or activation. Protein isoforms may have different
functions and Alms and White have cloned and expressed a naturally
occurring splice variant of IL-4, formed by the omission of exon 2,
thus called IL-4.delta.2..sup.86 It was observed that IL-4.delta.2
inhibits T-cell proliferation induced by IL-4.
However, the art lacks any knowledge about IL-9 protein isoforms
which are formed by deletions of exons 2 and 3 or the regulatory
functions exhibited by these truncated proteins. Specifically,
their role in regulating the biological activity, namely, the
down-regulation of IL-9 expression or activity is unclear.
Moreover, the formation of such isoforms by alternative splicing
has not been previously observed or used to provide variants of
IL-9 which function as agonists or antagonists of the native
cytokine.
The art also lacks any knowledge about the role of the IL-9
receptor with asthma-related disorders. It is known that IL-9 binds
to a specific receptor expressed on the surface of target
cells..sup.46, 52, 53 The receptor actually consists of two protein
chains: one protein chain, known as the IL-9 receptor, binds
specifically with IL-9 and the other protein chain is the chain,
which is shared in common with the IL-2 receptor..sup.46 In
addition, the human IL-9 receptor cDNA has been cloned..sup.46, 52,
53 This cDNA encodes a 522 amino acid protein which exhibits
significant homology to the murine IL-9 receptor. The extracellular
region of the receptor is highly conserved, with 67% homology
existing between the murine and human proteins. The cytoplasmic
region of the receptor is less highly conserved. The human
cytoplasmic domain is much larger than the corresponding region of
the murine receptor..sup.46
The IL-9 receptor gene has also been characterized..sup.53 It is
thought to exist as a single copy in the mouse genome and is
composed of nine exons and eight introns..sup.53 The human genome
contains at least four IL-9 receptor pseudogenes. The human IL-9
receptor gene has been mapped to the 320 kb subtelomeric region of
the sex chromosomes X and Y..sup.46 Nonetheless, despite these
studies, the art lacks any knowledge of a relation between the IL-9
receptor and atopic allergy, asthma, or bronchial
hyperresponsiveness.
Thus, the art lacks any knowledge of how the IL-9 gene, its
receptor, and their functions, are related to atopic allergy,
asthma, bronchial hyperresponsiveness, and related disorders.
Therefore, there is a specific need in the art for genetic
information on atopic allergy, asthma, bronchial
hyperresponsiveness, and for elucidation of the role of IL-9 in the
etiology of these disorders. There is also a need for elucidation
of the role of the IL-9 receptor and the IL-9 receptor gene in
these disorders. Furthermore, most significantly, based on this
knowledge, there is a need for the identification of agents which
are capable of regulating the interaction between IL-9 and its
receptor for treating these disorders.
SUMMARY OF THE INVENTION
Applicant has satisfied the long felt need for a treatment for
atopic allergy including asthma and related disorders by providing
information demonstrating the role of IL-9 (also known as Asthma
Associated Factor 1, or AAFI) in the pathogenesis of these
disorders which information has led to compounds that are capable
of regulating the activity of IL-9. Applicant has also demonstrated
conserved linkage and synteny homologies between mice and humans
for a gene that determines biologic variability in airway
hyperresponsiveness. These relationships specifically identify IL-9
as a gene candidate. In addition, applicant has determined that
IL-9 is critical to a number of antigen-induced responses in mice
including bronchial hyperresponsiveness, eosinophilia and elevated
cell counts in bronchial lavage, and elevated serum total IgE.
These findings typify the allergic inflammation associated with
asthma.
Furthermore, applicant has determined that a C to T nucleic acid
variation at position 3365 in exon 5 of the human IL-9 gene
produces the predicted amino acid substitution of a methionine for
a threonine at codon 117 of IL-9. When this substitution occurs in
both alleles in one individual, it is associated with less evidence
of atopic allergy including asthma, fewer abnormal skin test
responses, and a lower serum total IgE. Thus, applicant has
identified the existence of a nonasthmatic, nonatopic phenotype
characterized by methionine at codon 117 when it occurs in both
IL-9 gene products in one individual. As an additional significant
corollary, applicant has identified the existence of susceptibility
to an asthmatic, atopic phenotype characterized by a threonine at
codon 117. Thus, the invention includes purified and isolated DNA
molecules having such a sequence as well as the peptides encoded by
such DNA.
The biological activity of IL-9 results from its binding to the
IL-9 receptor and the consequent propagation of a regulatory signal
in specific cells. Therefore, IL-9 functions can be interrupted or
regulated by the interaction of IL-9 agonists or antagonists with
IL-9 or its receptor. Down regulation, i.e. reduction of the
functions controlled by IL-9, is achieved in a number of ways.
Administering agonists or antagonists that can interrupt the
binding of IL-9 to its receptor is one key mechanism and such
agonists and antagonists are within the claimed invention. Examples
include administration of polypeptide products encoded by the DNA
sequences of IL-9 or IL-9 receptor wherein the DNA sequences
contain various mutations. These mutations may be point mutations,
insertions, deletions, or spliced variants of IL-9 or its
receptor.
A further embodiment of this invention includes the regulation of
the activity of IL-9 by administering "agonists and antagonists."
The skilled artisan will readily recognize that all molecules
containing the requisite 3-dimensional structural conformation and
which contain the residues essential or critical for receptor
binding are within the scope of this invention. Specifically,
residues 43-60 and 71-90 of the mature protein appear to be
important for receptor binding. Applicant has shown that peptides
KP-16 (residues 43-60) and KP-20 (residues 71-90) act as receptor
antagonists. In addition, these residues in the native IL-9
molecule are predicted to form anti-parallel helical structures.
The three dimensional structure of the protein suggests that
specifically serine 52 and/or glutamic acid 53 interact with lysine
85, serine 56 interacts with lysine 82, and threonine 59 interacts
with valine 78. The three dimensional coordinates of these
anti-parallel helices and the related functional groups represent
the requisite 3-dimensional conformation critical for receptor
binding and compounds which simulate these relationships are within
the scope of this invention.
The biological activity of the IL-9 receptor (also called Asthma
Associates Factor 2, AAF2) can also be modulated by using soluble
IL-9 receptor molecules. Such a molecule prevents the binding of
IL-9 to the cell-bound receptor and acts as an antagonist for IL-9,
and is also within the scope of this invention.
Polyclonal and monoclonal antibodies which block the binding of
IL-9 to its receptor are also within the scope of this invention
and are useful therapeutic agents in treating atopic allergy
including asthma and related disorders.
Another embodiment of this invention relates to the use of isolated
DNA sequences containing various mutations such as point mutations,
insertions, deletions, or spliced mutations of IL-9 or the IL-9
receptor in gene therapy.
Expression of IL-9 and IL-9 receptor is also down-regulated by
administering an effective amount of synthetic antisense
oligonucleotide sequences. The oligonucleotide compounds of the
invention bind to the mRNA coding for human IL-9 and IL-9 receptor
thereby inhibiting expression of these molecules.
The structure of both IL-9 and the IL-9 receptor have been examined
and analyzed in great detail and amino acid residues of IL-9
critical for receptor binding have been identified. Based on
structural studies and the binding characteristics of this specific
binding pair, this invention further includes small molecules
tailored such that their structural conformation provides the
residues essential for blocking the interaction of IL-9 with the
IL-9 receptor. Such blockade results in modulation of the activity
of the receptor and these molecules are, therefore, useful in
treating atopic allergies.
Another embodiment of this invention is directed to the regulation
of downstream signaling pathways necessary for IL-9 function. IL-9
induces tyrosine phosphorylation of Stat3 which appears to be
unique to the IL-9 signaling pathway.sup.58 and is useful as a
target for inhibitors. Specific and nonspecific inhibitors of
tyrosine kinase such as tyrophostins are, therefore, useful in
downstream regulation of the physiological activity of IL-9, and
are part of the invention.
In a further embodiment aminosterol compounds are also useful in
treating atopic allergies and related disorders because they are
also involved in blocking signal transduction of the IL-9 signal
transduction pathway.
The products discussed above represent various effective
therapeutic agents in treating atopic allergies, asthma and other
related disorders.
This invention also includes the truncated polypeptides encoded by
the DNA molecules described above. These polypeptides are capable
of regulating the interaction of IL-9 with the IL-9 receptor.
Thus, applicant has identified the critical role of the IL-9
pathway in pathogenesis of atopic allergy, including bronchial
hyperresponsiveness, asthma, and related disorders. More
specifically, applicant has provided antagonists and methods of
identifying antagonists that are capable of regulating the
interaction between IL-9 and its receptor. Applicant also provides
methods for regulating the activity of IL-9 by: 1) administering a
compound having activity comparable to IL-9 containing methionine
at codon 117 and the ability to bind to a receptor for IL-9 in an
amount sufficient to down-regulate the activity of IL-9; and 2) by
administering truncated protein products encoded by isolated
nucleic acid sequences comprising deletions of any one or more of
exons 1, 2, 3, 4, or 5.
Having identified the critical role of the IL-9 pathway in atopic
allergy, bronchial hyperresponsiveness, and asthma, applicant also
provides a method for the diagnosis of susceptibility to atopic
allergy, asthma, and related disorders. Lastly, applicant provides
a method for assaying the functions of IL-9 and its receptor to
identify compounds or agents that may be administered in an amount
sufficient to down-regulate either the expression or functions of
IL-9 and the IL-9 receptor.
The accompanying figures, which are incorporated in and constitute
a part of this specification, illustrate several embodiments of the
invention and, together with the description, serve to explain the
principle of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Map showing the relative order and distance in centiMorgans
(cM) between the polymorphic genetic markers useful for mapping
asthma-related genes.
FIG. 2: Illustration of the genetic map of human chromosome
5q31-q33 and syntenic regions in the mouse.
FIG. 3: The LOD score curve on mouse chromosome 13 for
atracurium-induced airway responsiveness in mice with increased
susceptibility to bronchoconstrictor stimuli.
FIG. 4: Alignment of amino acid sequences corresponding to exon 5
of the human and murine IL-9 genes. The first sequence is
translated from the Thr allele of the human gene. The middle
sequence is translated from the Met allele of the human gene. The
final sequence is translated from the murine gene.
FIG. 5: Histogram of the correlation between human IL-9 gene
alleles and serum total IgE titers measured in international units.
S/S denotes Thr/Thr individuals, S/R denotes Thr/Met individuals
and R/R denotes Met/Met individuals.
FIG. 6: Illustration the simple sequence repeat polymorphism at the
IL-9 locus.
FIG. 7: Translated cDNA sequence of Thr117 version of IL-9.
FIG. 8: Translated cDNA sequence of Met117 version of IL-9.
FIG. 9: Map of pFlag expression construct with Thr117.
FIG. 10: Sequence of pFlag expression construct for the Thr117
version of the cDNA from the region surrounding the site of
ligation.
FIG. 11: Map of pFlag expression construct with Met117.
FIG. 12: Sequence of pFlag expression construct for the Met117
version of the cDNA from the region surrounding the site of
ligation.
FIG. 13: Western blot of recombinant IL-9 proteins
FIG. 14: Amino acid sequences for inhibitory peptides.
FIG. 15: Inhibition by KP-16 of IL-9 mediated MO7e
proliferation.
FIG. 16: Inhibition by KP-20 of IL-9 mediated MO7e
proliferation.
FIG. 17: Inhibition by KP-23 of IL-9 mediated MO7e
proliferation.
FIG. 18: Inhibition by various tyrophostins of IL-9 mediated MO7e
proliferation.
FIG. 19: Inhibition by various aminosterols of IL-9 mediated MO7e
proliferation.
FIGS. 20A and 20B: Characterization of the role of IL-9 in the
antigen response in vivo.
FIG. 21: Histologic examination of lungs from control, ova
challenged, and anti-IL-9 pretreated animals.
FIG. 22: Inhibition of the antigen response in vivo by blocking
antibodies to the murine IL-9 receptor.
FIG. 23: Expression of human Met117IL-9 and Thr117IL-9.
FIG. 24: Binding of the human recombinant Met117 and Thr117 forms
of IL-9 to a soluble receptor.
FIG. 25: Steady state levels of IL-9 in unstimulated and stimulated
murine splenocytes.
FIGS. 26A and 26B: An appendix of chemical moieties.
FIG. 27: Aminosterols isolated from the dog fish shark.
DETAILED DESCRIPTION OF THE INVENTION
Applicant has resolved the needs in the art by elucidating an IL-9
pathway and compositions that affect that pathway that may be used
in the diagnosis, prevention or treatment of atopic allergy
including asthma and related disorders. Asthma encompasses
inflammatory disorders of the airways with reversible airflow
obstruction. Atopic allergy refers to atopy, and related disorders
including asthma, bronchial hyperresponsiveness (BHR), rhinitis,
urticaria, allergic inflammatory disorders of the bowel, and
various forms of eczema. Atopy is a hypersensitivity to
environmental allergens expressed as the elevation of serum total
IgE or abnormal skin test responses to allergens as compared to
controls. BHR refers to bronchial hyperresponsiveness, a heightened
bronchoconstrictor response to a variety of stimuli.
By analyzing the DNA of families that exhibit asthma-related
disorders, applicant has identified a polymorphism in the IL-9 gene
that correlates with the biologic variability of serum total IgE as
one measurable expression of atopy. The IL-9 gene (also known as
Asthma Associated Factor 1 or AAF1) refers to the genetic locus of
interleukin-9, a cytokine exhibiting a variety of functions
involving the regulation of human myeloid and lymphoid systems. The
IL-9 gene of the present invention is found in the q31-q33 region
of human chromosome 5 and chromosome 13 in the mouse.
By polymorphism, applicant means a change in a specific DNA
sequence, termed a "locus", from the prevailing sequence. In
general, a locus is defined as polymorphic when artisans have
identified two or more alleles encompassing that locus and the
least common allele exists at a frequency of 1% or more.
The polymorphism of the present invention leads to an amino acid
substitution at residue 117 of IL-9. Specifically, instead of the
hydrophilic amino acid threonine, the IL-9 of the present invention
exhibits the hydrophobic amino acid methionine (Met IL-9). On a
genetic level, the polymorphism of the present invention is a
substitution of a thymine residue for a cytosine residue at
nucleotide position 3365 in the human IL-9 gene as it is described
by Renauld and colleagues (1990) (GenBank accession numbers M30135
and M30136),.sup.54 or at the comparable nucleotide position 4244
of the human IL-9 gene sequence reported by Kelleher et al., (1991)
(GenBank accession number M86593)..sup.55
Individuals with a threonine (Thr) at amino acid 117 of IL-9 in
either one or both of their alleles (Thr/Thr or Thr/Met) generally
exhibit susceptibility to an asthmatic or atopic allergic
phenotype, and these genotypes are characterized by higher mean
serum total IgE levels in the populations studied. In contrast,
those individuals with a methionine (Met) at codon 117 of IL-9 in
both alleles (Met/Met) exhibit a lack of asthma, fewer abnormal
skin test responses, and a lower serum total IgE. Thus, the Met/Met
genotype of IL-9 appears to protect against asthma or atopic
allergy.
Accordingly, the invention provides a purified and isolated DNA
molecule comprising a nucleotide sequence encoding human
interleukin 9 containing methionine at position 117 (Met IL-9), or
a fragment thereof. The invention also includes degenerate
sequences of the DNA as well as sequences that are substantially
homologous. The source of the IL-9 of the invention is human.
Alternatively, the DNA or fragment thereof may be synthesized using
methods known in the art. It is also possible to produce the
compound by genetic engineering techniques, by constructing DNA by
any accepted technique, cloning the DNA in an expression vehicle
and transfecting the vehicle into a cell which will express the
compound. See, for example, the methods set forth in Sambrook et
al., (1985) MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. Cold
Spring Harbor Laboratory Press.
Airway hyperresponsiveness is found in virtually all asthmatics and
in some strains of inbred mice (DBA/2)..sup.84 Airway
hyperresponsiveness is a risk factor for the development of asthma
in humans and is used in animal models of asthma as a physiologic
measure to assess the efficacy of treatment for asthma. These data
along with human.sup.79 and murine genetic mapping results (see
Examples 1 and 2) suggest a critical role for the murine IL-9 gene
product in the airway response of the mouse. In particular, the
hyperresponsive DBA/2(D2) mice differ from the
C57BL/6(B6)hyporesponsive mice.sup.84 in their expression of steady
state levels of IL-9 (See Example 14, FIG. 26). Furthermore,
pretreatment with blocking antibodies to IL-9/IL-9 receptor can
optionally provide complete protection from antigen induced airway
hyperresponsiveness and inflammation in mice demonstrating a
critical regulatory role for IL-9 in these immune responses. Thus,
these data demonstrate that although different molecular changes
produce biologic variability in airway responsiveness in humans and
mice, these changes arise in the same gene(s) (IL-9/IL-9R) that
regulate this pathway. Taken together, these observations confirm
the critical role of IL-9 and the IL-9 receptor in airway
hyperresponsiveness, asthma, and atopic allergy. Moreover, these
data demonstrate that agents of the convention, which block the
interaction of IL-9 with its receptor, protect against an antigen
induced response such as those detailed above.
Further evidence defining the critical role of IL-9 in the
pathogenesis of atopic allergy, bronchial hyperresponsivenss,
asthma, and related disorders derives directly from the applicants
observation that IL-9 is critical to a number of antigen induced
responses in mice. When the functions of IL-9 are down regulated by
antibody pretreatment prior to aerosol challenge with antigen, the
animals can be completely protected from the antigen induced
responses. These responses include: bronchial hyperresponsiveness,
eosinophilia and elevated cell counts in bronchial lavage,
histologic changes in lung associated with inflammation, and
elevated serum total IgE. Thus, the treatment of such responses,
which are critical to the pathogenesis of atopic allergy and which
characterize the allergic inflammation associated with asthma, by
the down regulation of the functions of IL-9, are within the scope
of this invention.
Applicant also teaches the regulation of the activity of IL-9 by
administering "agonists and antagonists" to the IL-9 receptor. The
skilled artisan will readily recognize that all molecules
containing the requisite 3-dimensional structural conformation and
which contain the residues essential or critical for receptor
binding are within the scope of this invention. Applicant has shown
that peptides KP-16 (IL-9 residues 43-60) and KP-20 (IL-9 residues
71-90) (produced using standard peptide automated synthesis
techniques, for example, the Applied Biosystems Model 431A Peptide
Synthesizer) act as IL-9 antagonists. Specifically, applicant
demonstrates that residues 43-60 and 71-90 of the mature protein
appear to be important for receptor binding. In addition, these
residues include most of exon 4 (amino acids 44-88) and are
predicted to form anti-parallel helical structures. The three
dimensional structure of the protein suggests that specifically
serine 52 and/or glutamic acid 53 interact with lysine 85, serine
56 interacts with lysine 82, and threonine 59 interacts with valine
78. The three dimensional coordinates of these parallel helices and
the related functional groups represent the requisite 3-dimensional
conformation critical for receptor binding and compounds that
simulate these relationships are within the scope of this
invention.
The demonstration of an IL-9 sequence associated with an
asthma-like phenotype, and one associated with the lack of an
asthma-like phenotype, indicates that the lungs' inflammatory
response to antigen is dependent on IL-9, and therefore, that down
regulating the function of IL-9 should protect against the antigen
induced response. Furthermore, applicant also provides methods of
diagnosing susceptibility to atopic allergy and related disorders
and for treating these disorders based on the relationship between
IL-9 and its receptor.
A receptor is a soluble or membrane bound component that recognizes
and binds to molecules, and the IL-9 receptor (also known as Asthma
Associated Factor 2 or AAF2) of the invention is the component that
recognizes and binds to IL-9. The functions of the IL-9 receptor
consist of binding an IL-9-like molecule and propagating its
regulatory signal in specific cells..sup.57-60 An interruption of
that function will lead to a down regulation, i.e., reduction, of
either the expression of IL-9 or of the functions controlled by
IL-9. Accordingly, by virtue of this interaction between IL-9 and
the IL-9 receptor, certain functions of the organism are modulated
or controlled. For a general discussion of receptors, see Goodman
and Gilman's The Pharmacologic Basis of Therapeutics, 7th Edition,
MacMillan Publishing Company.
One diagnostic embodiment involves the recognition of variations in
the DNA sequence of IL-9. One method involves the introduction of a
nucleic acid molecule (also known as a probe) having a sequence
complementary to the IL-9 of the invention under sufficient
hybridizing conditions, as would be understood by those in the art.
In one embodiment, the sequence will bind specifically to the
Met117 IL-9 or to Thr117 IL-9, and in another embodiment will bind
to both Met117 IL-9 and Thr117 IL-9. Another method of recognizing
DNA sequence variation associated with these disorders is direct
DNA sequence analysis by multiple methods well known in the
art..sup.77 Another embodiment involves the detection of DNA
sequence variation in the IL-9 gene associated with these
disorders..sup.73-77 These include the polymerase chain reaction,
restriction fragment length polymorphism (RFLP) analysis and single
stranded conformational analysis. In a preferred embodiment,
applicant provides specifically for a method to recognize, on a
genetic level, the polymorphism in IL-9 associated with the Thr and
Met alleles using a StyI RFLP as described herein. In other
embodiments Nla, Pfim1, PflM1, and Nco1 RFLPs may be used to
distinguish these two alleles of IL-9 genes.
Another embodiment involves treatment of atopic allergy and related
disorders. In a preferred embodiment, the applicant provides a
method of administering a compound having activity comparable to
Met IL-9 and the ability to bind to an IL-9 receptor in an amount
sufficient to down regulate the activity of IL-9. A compound having
activity comparable to Met IL-9 is a compound that functions
similarly but not necessarily identically. Thus, it may bind to the
IL-9 receptor but without the same physiological effects. Examples
include amino acid sequences of IL-9 containing various point
mutations and/or deletions and sequences substantially homologous
thereto. For example, such a compound may interrupt the binding of
Thr IL-9 to the IL-9 receptor as measured by techniques known in
the art. The invention also encompasses functionally effective
fragments of the above amino acid sequences. In one such technique,
the Thr IL-9 may be considered a "ligand" for the IL-9 receptor,
and binding between the two may be assessed by ligand-binding
assays which are well known in the art as set forth in Goodman and
Gilman's The Pharmacologic Basis of Therapeutics, 7th Edition,
MacMillan Publishing Company.
In another embodiment, the compound may resemble the Met allele of
IL-9 in structure. Thus, such a compound may incorporate a
methionine in codon 117 of IL-9 or may incorporate another
hydrophobic amino acid. Thus, included within the scope of this
invention are IL-9 varients comprising substitutions of Thr at
position 117 by amino acids selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, phenylalanine,
tryptophan, and methionine. Alternatively, the compound of the
invention may exist as a fragment of IL-9 with a structural
composition similar to Met IL-9. In another embodiment of the
invention, the compound may retain functions comparable to Met
IL-9, but may not resemble Met IL-9 in structure. For example, the
composition of the compound may include molecules other than amino
acids. This example is merely illustrative and one of ordinary
skill in the art would readily recognize that other substitutions
and/or deletion analogs of IL-9 resulting in effective antagonists
are also within the scope of this invention. As discussed above all
molecules containing the requisite 3-dimensional structural
conformation and which contain the residues essential or critical
for receptor binding are within the scope of this invention.
Specific assays may be based on IL-9's known regulation, in part,
of the proliferation of T lymphocytes, IgE synthesis, and release
from mast cells..sup.54-60 Another assay involves the ability of
human IL-9 to specifically induce the rapid and transient tyrosine
phosphorylation of multiple proteins in M07e cells..sup.57 Because
this response is dependent on the expression and activation of the
IL-9 receptor, it represents a simple method or assay for the
characterization of potentially valuable compounds. The tyrosine
phosphorylation of Stat3 transcriptional factor appears to be
specifically related to the actions of IL-9,.sup.58 and this
response represents a simple method or assay for the
characterization of compounds within the invention. Still another
method to characterize the function of IL-9 and IL-9-like molecules
involves the well known murine TS1 clone and the D10 clone
available from ATCC used to assess human IL-9 function with a
cellular proliferation assay..sup.59 The Met IL-9 that forms a part
of the invention may be viewed as a "weak agonist" of the IL-9
receptor. Such weak agonists are another preferred embodiment of
the invention. The term agonist, according to this invention,
includes compounds that mimic at least some of the effects of
endogenous compounds by interacting or binding with a receptor.
Agonists that interact or bind to the IL-9 receptor on the surface
of certain cells initiate a series of biochemical and physiological
changes that are characteristic of this cytokine's
actions..sup.45-5, 54-60 To identify other weak agonists of the
invention, one may test for binding to the IL-9 receptor or for
IL-9-like functions as described herein and in the cited
literature..sup.2, 45-51, 54-60
The present invention also includes antagonists of IL-9 and its
receptor. Antagonists are compounds that are themselves devoid of
pharmacological activity but cause effects by preventing the action
of an agonist. To identify an antagonist of the invention, one may
test for competitive binding with a known agonist or for
down-regulation of IL-9-like functions as described herein and in
the cited literature..sup.2, 45-51, 54-60
The binding of either the agonist or antagonist may involve all
known types of interactions including ionic forces, hydrogen
bonding, hydrophobic interactions, van der Waals forces, and
covalent bonds. In many cases, bonds of multiple types are
important in the interaction of an agonist or antagonist with a
receptor.
In a further embodiment, these compounds may be analogs of IL-9.
IL-9 analogs may be produced by point mutations in the isolated DNA
sequence for the gene, nucleotide substitutions, and/or deletions
which can be created by methods that are all well described in the
art..sup.62 This invention also includes spliced variants of IL-9
and discloses isolated nucleic acid sequences of IL-9, which
contain deletions of one or more of its five exons. The term
"spliced variants" as used herein denotes a purified and isolated
DNA molecule encoding human IL-9 comprising at least one exon.
There is no evidence of naturally expressed spliced mutants in the
art. Thus, the present invention provides an isolated nucleic acid
containing exons 1, 4 and 5 of human IL-9. Other variants within
the scope of this invention include sequences comprising exons 1,
2, 3, 4, and 5; exons 1, 2, 3, and 4; exons 1, 2, 4, and 5 and
exons 1, 3, 4, and 5. It must be understood that these exons may
contain various point mutations.
Specific examples of antagonistic peptides derived from IL-9
include KP-16 (SEQ. ID NO: 13) and KP-20 (SEQ. ID NO: 14) which are
derived from exon 4. Exon 4 encodes 44 amino acids while the
peptides mentioned above contain 16 and 20 amino acids respectively
and they do not overlap. These peptides exhibit considerable
inhibitory activity both individually and when assayed in
combination. Additionally, KP-23 (SEQ ID NO: 15) and KP-24 (SEQ ID
NO: 16) are derived from exon 5 and also exhibit similar activity.
Splice variants of IL-9 can be formed by deletion of any one or
more of the IL-9 exons 1 through 5. As shown above, peptides
derived from these exons show regulatory capability and,
accordingly, are useful in treating atopic allergies, including
asthma.
It is known in the art that, in multienzyme systems, the first or
regulatory enzyme can be activated or inhibited by the end product
of the multi-enzyme system. When the concentration of the end
product increases over the steady state concentration, the end
product will act as a specific activator or inhibitor of the
regulatory enzyme in the sequence. Such feedback mechanism is also
relevant to the IL-9 system and it is observed that the various
polypeptides of this invention are capable of exerting such
activation or inhibitory control on the activity of the IL-9
receptor and possibly the expression or function of other cytokines
and their receptors that play a role in the pathogenesis of
asthma.
The invention also includes modifications of agonists or
antagonists that can be made using knowledge that is routine to
those in this art. For example, the affinity of a compound for a
receptor is generally closely related to the chemical structure of
the compound. Thus, structure-activity relationships may be used to
modify the agonists and antagonists of the invention. For example,
the techniques of crystallography/X-ray diffraction and NMR may be
used to make modifications of the invention.
For example, one can create a three dimensional structure of human
IL-9 that can be used as a template for building structural models
of deletion mutants using molecular graphics. These models can then
be used to identify and construct a mutant: IL-9 molecule with
affinity for the IL-9 receptor comparable to IL-9, but with a lower
biologic activity. What is meant by lower biologic activity is 2 to
100,000 fold less than IL-9, preferably 100 to 1,000 fold less than
IL-9.
In still another embodiment, these compounds also may be used as
dynamic probes for receptor structure and to develop receptor
antagonists using IL-9 dependent cell lines.
In addition, this invention also provides compounds that prevent
the synthesis or reduce the biologic stability of IL-9 or the IL-9
receptor. Biologic stability is a measure of the time between the
synthesis of the molecule and its degradation. For example, the
stability of a protein, peptide or peptide mimetic.sup.89
therapeutic may be prolonged by using D-amino acids, or shortened
by altering its sequence to make it more susceptible to enzymatic
degradation.
In another embodiment, the agonists and antagonists of the
invention are antibodies to IL-9 and the IL-9 receptor. The
antibodies to IL-9 and its receptor may be either monoclonal or
polyclonal made using standard techniques well known in the art
(See Harlow & Lane (1988) Antibodies--A Laboratory Manual,
(Cold Spring Harbor Laboratory, Press. They can be used to block
IL-9 from binding to the receptor. In one embodiment the antibodies
interact with IL-9. In another embodiment the antibodies interact
with the IL-9 receptor. The IL-9 used to elicit these antibodies
can be any of the IL-9 varients discussed above.
Antibodies are also produced from peptide sequences of IL-9 or the
IL-9 receptor using standard techniques in the art (see Protocols
in Immunology, Chapt. 9, Wiley). The peptide sequences from the
murine IL-9 receptor that can be used to produce blocking antisera
have been identified as: GGQKAGAFTC (residues 1-10)(SEQ ID NO:19);
LSNSIYRIDCHWSAPELGQESR (residues 11-32)(SEQ ID NO:20); and
CESYEDKTEGEYYKSHWSEWS (residues 184-203 with a Cys residue added to
the N-terminus for coupling the peptide to the carrier protein)(SEQ
ID NO:21). In addition, an epitope that binds to a blocking
antibody directed to the human IL-9 receptor has been identified as
residues 8-14 of the mature human IL-9 receptor. (TCLTNNI)(SEQ ID
NO:22) and two epitopes that bind to blocking antibodies directed
to human IL-9 have also been identified as residues 50-67
(CFSERLSQMTNTTMQTRY) (SEQ ID NO:23) and residues 99-116
(TAGNALTFLKSLLEIFQK) (SEQ ID NO:16) The human epitopes as well as
the human peptides that correspond to the peptides that produce
blocking antibodies in the murine sequences are most likely to be
useful for the production of therapeutic antibodies.
In still another embodiment, the compounds of the invention may be
coupled to chemical moieties, including proteins that alter the
functions or regulation of the IL-9 pathway for therapeutic benefit
in atopic allergy and asthma..sup.61 These proteins may include in
combination other cytokines and growth factors including.sup.67
L-4, IL-5, IL-3, IL-2, IL-13, and IL-10 that may offer additional
therapeutic benefit in atopic allergy and asthma. In addition, the
IL-9 of the invention may also be conjugated through
phosphorylation and conjugated to biotinylate, thioate, acetylate,
iodinate, and any of the crosslinking reagents shown in FIGURES 26A
and B (Pierce).
In a further embodiment, the invention includes the down regulation
of IL-9 expression or function by administering soluble IL-9
receptor molecules that bind IL-9. Renauld et al., (1992).sup.59
have shown the existence of a soluble form of the IL-9 receptor.
This molecule can be used to prevent the binding of IL-9 to cell
bound receptor and act as an antagonist of IL-9. Soluble receptors
have been used to bind cytokines or other ligands to regulate their
function..sup.87 A soluble receptor is a form of a membrane bound
receptor that occurs in solution, or outside of the membrane.
Soluble receptors may occur because the segment of the molecule
which commonly associates with the membrane is absent. This segment
is commonly referred to in the art as the transmembrane domain of
the gene, or membrane binding segment of the protein. Thus, in one
embodiment of the invention, a soluble receptor may represent a
fragment or an analog of a membrane bound receptor. In another
embodiment of the invention, the structure of the segment that
associates with the membrane may be modified (e.g. DNA sequence
polymorphism or mutation in the gene) so the receptor is not
inserted into the membrane, or the receptor is inserted, but is not
retained within the membrane. Thus, a soluble receptor, in contrast
to the corresponding membrane bound form, differs in one or more
segments of the gene or receptor protein that are important to its
association with the membrane..sup.52, 53
These compounds may be known forms of a soluble IL-9 receptor that
act to bind IL-9. Alternatively, these compounds may resemble known
forms of the IL-9 receptor, but may exist as fragments. In another
embodiment of the invention, the compound may retain functions
comparable to soluble IL-9 receptor, but may not resemble soluble
IL-9 receptor in composition. For example, the composition of the
compound may include molecules other than amino acids. Thus, these
compounds will bind IL-9 and prevent IL-9 from acting at its cell
surface receptor.
A further embodiment of the invention relates to antisense or gene
therapy. It is now known in the art that altered DNA, molecules can
be tailored to provide a specific selected effect, when provided as
antisense or gene therapy. The native DNA segment coding for IL-9
receptor, has, as do all other mammalian DNA strands, two strands;
a sense strand and an antisense strand held together by hydrogen
bonding. The mRNA coding for the receptor has a nucleotide sequence
identical to the sense strand, with the expected substitution of
thymidine by uridine. Thus, based upon the knowledge of the
receptor sequence, synthetic oligonucleotides can be synthesized.
These oligonucleotides can bind to the DNA and RNA coding for the
receptor. The active fragments of the invention, which are
complementary to mRNA and the coding strand of DNA, are usually at
least about 15 nucleotides, more usually at least 20 nucleotides,
preferably 30 nucleotides and more preferably may be 50 nucleotides
or more. The binding strength between the sense and antisense
strands is dependent upon the total hydrogen bonds. Therefore,
based upon the total number of bases in the mRNA, the optimal
length of the oligonucleotide sequence may be easily calculated by
the skilled artisan.
The sequence may be complementary to any portion of the sequence of
the mRNA, i.e., it may be proximal to the 5'-terminus or capping
site, or downstream from the capping site, between the capping site
and the initiation codon and may cover all or only a portion of the
non-coding region or the coding region. The particular site(s) to
which the antisense sequence binds will vary depending upon the
degree of inhibition desired, the uniqueness of the sequence, the
stability of the antisense sequence, etc.
In the practice of the invention, expression of the IL-9 receptor
is down-regulated by administering an effective amount of synthetic
antisense oligonucleotide sequences described above. The
oligonucleotide compounds of the invention bind to the mRNA coding
for human IL-9 or IL-9 receptors thereby inhibiting expression
(translation) of these proteins. (see Gruss et al., (1992) Cancer
Res. 52, 1026-1031).
The isolated DNA sequences containing various mutations such as
point mutations, insertions, deletions, or spliced mutations of
IL-9 are useful in gene therapy as well.
In addition to the direct regulation of the IL-9 receptor, this
invention also encompasses methods of downstream regulation which
involve inhibition of signal transduction. In particular, a further
embodiment of this invention is drawn to inhibition of tyrosine
phosphorylation. It is known in the art that highly exergonic
phosphoryl-transfer reactions are catalyzed by various enzymes
known as kinases. In other words, a kinase transfers phosphoryl
groups between ATP and a metabolite. IL-9 induces tyrosine
phosphorylation of multiple proteins; it is known in the art that
in addition to the activation of JAK1 and JAK3 tyrosine kinases,
IL-9 also induces tyrosine phosphorylation of Stat3..sup.58
Phoshorylation of Stat3 is unique to the IL-9 signal transduction
pathway and hence is a perfect target for inhibitors..sup.58 This
invention includes within its scope tyrphostins which are specific
inhibitors of protein tyrosine kinases. Thus, tyrphostins (obtained
for example from Calbiochem) and other similar inhibitors of these
kinases are useful in the modulation of signal transduction and are
useful in the treatment of atopic allergies and asthma.
In still another aspect of the invention, it was surprisingly,
found that aminosterol compounds are also useful in the inhibition
of signal transduction due to IL-9 stimulation. Aminosterol
compounds which are useful in this invention are described in U.S.
patent Ser. No. 5,637,691 and related U.S. patents Ser. Nos.
5,733,899 and 5,721,226, as well as in 5,840,740 and related U.S.
patent Ser. Nos. 5,795,885; 5,994,336; 5,763,430; 5,840,936;
5,874,597; 5,792,635 and 5,847,172 which are specifically
incorporated herein by reference.
In addition, the invention includes pharmaceutical compositions
comprising the compounds of the invention together with a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water is a preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectionable solutions. Suitable pharmaceutical
carriers are described in Remington's Pharmaceutical Sciences,
specifically incorporated herein by reference.
The compounds used in the method of treatment of this invention may
be administered systemically or topically, depending on such
considerations as the condition to be treated, need for
site-specific treatment, quantity of drug to be administered, and
similar considerations.
Topical administration may be used. Any common topical formation
such as a solution, suspension, gel, ointment, or salve and the
like may be employed. Preparation of such topical formulations as
are well described in the art of pharmaceutical formulations as
exemplified, for example, by Remington's Pharmaceutical Science,
Edition 17, Mack Publishing Company, Easton, Pa. For topical
application, these compounds could also be administered as a powder
or spray, particularly in aerosol form. The active ingredient may
be administered in pharmaceutical compositions adapted for systemic
administration. As is known, if a drug is to be a administered
systemically, it may be confected as a powder, pill, tablets or the
like, or as a syrup or elixir for oral administration. For
intravenous, intraperitoneal or intra-lesional administration, the
compound will be prepared as a solution or suspension capable of
being administered by injection. In certain cases, it may be useful
to formulate these compounds in suppository form or as an extended
release formulation for deposit under the skin or intermuscular
injection. In a preferred embodiment, the compounds of this
invention be administered by inhalation. For inhalation therapy the
compound may be in a solution useful for administration by metered
dose inhalers, or in a form suitable for a dry powder inhaler.
An effective amount is that amount which will down regulate either
the expression of IL-9 or the functions controlled by IL-9. A given
effective amount will vary from condition to condition and in
certain instances may vary with the severity of the condition being
treated and the patient's susceptibility to treatment. Accordingly,
a given effective amount will be best determined at the time and
place through routine experimentation. However, it is anticipated
that in the treatment of asthma-related disorders in accordance
with the present invention, a formulation containing between 0.001
and 5 percent by weight, preferably about 0.01 to 1%, will usually
constitute a therapeutically effective amount. When administered
systemically, an amount between 0.01 and 100 mg per kg body weight
per day, but preferably about 0.1 to 10 mg/kg, will effect a
therapeutic result in most instances.
Applicant also provides for a method to screen for the compounds
that down regulate the expression of IL-9 or the functions
controlled by IL-9. One may determine whether the functions
expressed by IL-9 are down-regulated using techniques standard in
the art..sup.57-60 In a specific embodiment, applicant provides for
a method of identifying compounds with functions comparable to Met
IL-9. Thus, in one embodiment, serum total IgE may be measured
using techniques well known in the art.sup.42 to assess the
efficacy of a compound in down regulating the functions of IL-9 in
vivo. In another embodiment, bronchial hyperresponsiveness,
bronchoalveolar lavage, and eosinophilia may be measured using
techniques well known in the art.sup.42 to assess the efficacy of a
compound in down regulating the functions of IL-9 in vivo. In yet
another embodiment, the functions of IL-9 may be assessed in vitro.
As is known to those in the art, human IL-9 specifically induces
the rapid and transient tyrosine phosphorylation of multiple
proteins in M07e cells. The tyrosine phosphorylation of Stat3
transcriptional factor appears to be specifically related to the
actions of IL-9. Another method to characterize the function of
IL-9 and IL-9-like molecules that depends on the "stable
expression" of the IL-9 receptor uses the well known murine TS1
clones to assess human IL-9 function with a cellular proliferation
assay..sup.59
The invention also includes a simple screening assay for saturable
and specific ligand binding based on cell lines that express the
IL-9 receptor..sup.46, 59 The IL-9 receptor is expressed in on a
wide variety of cell types, including K562, C8166-45, B cells, T
cells, mast cells, neutrophils, megakaryocytes (UT-7 cells),.sup.53
the human megakaryoblastic leukemia cell lines MO7e.sup.57,
TF1,.sup.59 macrophages, fetal thymocytes, the human kidney cell
line 293,.sup.53 and murine embryonic hippocampal progenitor cell
lines..sup.46, 52, 53 In another embodiment, soluble IL-9 receptor
may be used to evaluate ligand binding and potential receptor
antagonists.
The practice of the present invention will employ the conventional
terms and techniques of molecular biology, pharmacology,
immunology, and biochemistry that are within the ordinary skill of
those in the art (see, for example, Sambrook et al., (1985)
MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. Cold Spring Harbor
Laboratory Press, or Ausubel et al., (1994) CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons.
Nonetheless, we offer the following basic background information.
The body's genetic material, or DNA, is arranged on 46 chromosomes,
which each comprises two arms joined by a centromere. Each
chromosome is divided into segments designated p or q. The symbol p
is used to identify the short arm of a chromosome, as measured from
the centromere to the nearest telomere. The long arm of a
chromosome is designated by the symbol q. Location on a chromosome
is provided by the chromosome's number (i.e., chromosome 5) as well
as the coordinates of the p or q region (i.e., q31-q33). In
addition, the body bears the sex chromosomes, X and Y. During
meiosis, the X and Y chromosomes exchange DNA sequence information
in areas known as the pseudoautosomal regions.
DNA, deoxyribonucleic acid, consists of two complementary strands
of nucleotides, which include the four different base compounds,
adenine (A), thymine (T), cytosine (C), and guanine (G). A of one
strand bonds with T of the other strand while C of one strand bonds
to G of the other to form complementary "base pairs," each pair
having one base in each strand.
A sequential grouping of three nucleotides (a "codon") codes for
one amino acid. Thus, for example, the three nucleotides CAG codes
for the amino acid Glutamine. The 20 naturally occurring amino
acids, and their one letter codes, are as follows:
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D
Asparagine or Asx B Aspartic acid Cysteine Cys C Glutamine Gln Q
Glutamine Acid Glu E Glutamine or Glx Z Glutamic acid Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
Amino acids comprise proteins. Amino acids may be hydrophilic,
i.e., displaying an affinity for water, or hydrophobic, i.e.,
having an aversion to water. Thus, the amino acids designated as G,
A, V, L, I, P, F, Y, W, C and M are hydrophobic and the amino acids
designated as S, Q, K, R, H, D, E, N and T are hydrophilic. In
general, the hydrophilic or hydrophobic nature of amino acids
affects the folding of a peptide chain, and consequently the three
dimensional structure of a protein.
DNA is related to protein as follows: ##STR1##
Genomic DNA comprises all the DNA sequences found in an organism's
cell. It is "transcribed" into messenger RNA ("mRNA").
Complementary DNA ("cDNA") is a complementary copy of mRNA made by
reverse transcription of mRNA. Unlike genomic DNA, both mRNA and
cDNA contain only the protein-encoding or polypeptide-encoding
regions of the DNA, the so-called "exons." Genomic DNA may also
include "introns," which do not encode proteins.
In fact, eukaryotic genes are discontinuous with proteins encoded
by them, consisting of exons interrupted by introns. After
transcription into RNA, the introns are removed by splicing to
generate the mature messenger RNA (mRNA). The splice points between
exons are typically determined by consensus sequences that act as
signals for the splicing process. Splicing consists of a deletion
of the intron from the primary RNA transcript and a joining or
fusion of the ends of the remaining RNA on either side of the
excised intron. Presence or absence of introns, the composition of
introns, and number of introns per gene, may vary among strains of
the same species, and among species having the same basic
functional gene. Although in most cases, introns are assumed to be
nonessential and benign, their categorization is not absolute. For
example, an intron of one gene can represent an exon of another. In
some cases, alternate or different patterns of splicing can
generate different proteins from the same single stretch of DNA. In
fact, structural features of introns and the underlying splicing
mechanisms form the basis for classification of different kinds of
introns.
As to the exons, these can correspond to discrete domains or
motifs, as for example, functional domains, folding regions, or
structural elements of a protein; or to short polypeptide
sequences, such as reverse turns, loops, glycosylation signals and
other signal sequences, or unstructured polypeptide linker regions.
The exon modules of the present combinatorial method can comprise
nucleic acid sequences corresponding to naturally occurring exon
sequences or naturally occurring exon sequences which have been
mutated (e.g. point mutations, truncations, fusions).
Returning now to the manipulation of DNA, DNA can be cut, spliced,
and otherwise manipulated using "restriction enzymes" that cut DNA
at certain known sites and DNA ligases that join DNA. Such
techniques are well known to those of ordinary skill in the art, as
set forth in texts such as Sambrook, et al., (1985) MOLECULAR
CLONING: A LABORATORY MANUAL, 2d ed. Cold Spring Harbor Laboratory
Press or Ausubel et al., (1994) CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons.
DNA of a specific size and sequence can then be inserted into a
"replicon," which is any genetic element, such as a plasmid,
cosmid, or virus, that is capable of replication under its own
control. A "recombinant vector" or "expression vector" is a
replicon into which a DNA segment is inserted so as to allow for
expression of the DNA, i.e., production of the protein encoded by
the DNA. Expression vectors may be constructed in the laboratory,
obtained from other laboratories, or purchased from commercial
sources.
The recombinant vector (known by various terms in the art) may be
introduced into a host by a process generically known as
"transformation." Transformation means the transfer of an exogenous
DNA segment by any of a number of methods, including infection,
direct uptake, transduction, F-mating, microinjection, or
electroporation into a host cell.
Unicellular host cells, known variously as recombinant host cells,
cells, and cell culture, includebacteria, yeast, insect cells,
plant cells, mammalian cells and human cells. In particularly
preferred embodiments, the host cells include E.coli, Pseudonas,
Bacillis, Streptomyces, Yeast, CHO, R1-1, B-W, LH, COS-J, COS-7,
BSC1, BSC40, BMT10, and S69 cells. Yeast cells especially include
Saccharomyces, Pichia, Candida, Hansenula, and Torulopis.
As those skilled in the art recognize, the expression of the DNA
segment by the host cell requires the appropriate regulatory
sequences or elements. The regulatory sequences vary according to
the host cell employed, but include, for example, in prokaryotes, a
promoter, ribosomal binding site, and/or a transcription
termination site. In eukaryotes, such regulatory sequences include
a promoter and/or a transcription termination site. As those in the
art well recognized, expression of the polypeptide may be enhanced,
i.e., increased over the standard levels, by careful selection and
placement of these regulatory sequences.
In other embodiments, promoters that may be used include the human
cytomegalovierus (CMV) promoter, tetracycline inducible promoter,
simian virus (SV40) promoter, moloney murine leukemia long terminal
repeat (LTR) promoter, glucocorticoid inducible murine mammary
tumor virus (MMTV) promoter, Herpes thymidine kinase promoter,
murine and human .beta.-actin promoters, HTLV1 and HIV IL-9 5'
flanking region, human and mouse IL-9 receptor 5' flanking region,
bacterial tac promoter and drosophila heat shock scaffold
attachment region (SAR) enhancer elements.
The DNA may be expressed as a polypeptide of any length such as
peptides, oligopeptides, and proteins. Polypeptides also include
translational modifications such as glycosylations, acetylations,
phosphorylations, and the like.
Another molecular biologic technique of interest to the present
invention is "linkage analysis." Linkage analysis is an analytic
method used to identify the chromosome or chromosomal region that
correlates with a trait or disorder..sup.44 Chromosomes are the
basic units of inheritance on which genes are organized. In
addition to genes, artisans have identified "DNA markers" on
chromosomes. DNA markers are known sequences of DNA whose identity
and sequence can be readily determined. Linkage analysis
methodology has been applied to the mapping of disease genes, for
example, genes relating to susceptibility to asthma, to specific
chromosomes..sup.42, 44
Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed. It is intended that the
specifications and examples be considered exemplary only with a
true scope of the invention being indicated by the claims.
EXAMPLES
In conducting the experiments described in the Examples below,
applicant used the following methods:
Patient Populations
Asthma families were recruited from two sources..sup.27, 42, 79-83,
88 In each case patients were genotyped with respect to the
polymorphism at position 3365 of the human IL-9 gene (GenBank
accession number M30136).
A third population of 74 individuals was ascertained randomly with
respect to asthma and atopy from the East Coast of the United
States. The frequency of the Met substitution at codon 117 was used
as an unbiased estimate of the prevalence of this variant in the
general population.
A fourth population of 49 individuals was ascertained randomly with
respect to asthma and atopy from the Philadelphia, PA area. Total
serum IgE were assayed by enzyme-linked immunosorbent test (ELISA,
Genzyme, Cambridge, MA). DNA was extracted from the WBC in
peripheral blood from each individual. Analyses of genetic markers
(genotyping) and candidate genes were performed on the genomic DNA
extracted. Once again, the frequency of the Met substitution at
codon 117 was used as an unbiased estimate of the prevalence of
this variant in the general population.
Oligonucleotide Primers
All primers were designed using OLIGO 4.0. Characterization of the
IL-9 gene was carried out using primers surrounding each of the 5
exons of the reported sequence. The primer sequences surrounding
each exon were: exon 1 (upper) (5' GCT CCA GTC CGC TGT CAA 3' (SEQ
ID NO: 32) and (lower) (5' CTC CCC CTG CAG CCT ACC 3' (SEQ ID NO:
33) (product size 150 bp; exon 2 (upper) (5' CGG GGC TGA CTA AAG
GTT CT 3' (SEQ ID NO: 34) and (lower) (5' GTT CTT AAA GAG CAT TCA
CT 3'(SEQ ID NO: 35) (product size 99 bp; exon 3 (upper) (5' ATT
TTC ACA TCT GGA ATC TTC ACT 3'(SEQ ID NO: 36) and (lower) (5' AAT
CCA AGG TCA ACA TTA TG 3' (SEQ ID NO: 37) (product size 113 bp;
exon 4 (upper) (5' TTT CTT TGA ATA AAT CCT TAC 3' (SEQ ID NO: 38)
and (lower) (5' GAA ATC ACC AAC AGG AAC ATA 3' (SEQ ID NO: 39)
(product size 206 bp; and exon 5 (upper) (5'ATC AAC TTT CAT CCC CAC
AGT 3' (SEQ ID NO: 40) and (lower) (5' GGA TAA ATA ATA TTT CAT CTT
CAT 3' (SEQ ID NO: 41). Each exon was examined first by a single
strand conformational polymorphism assay (SSCP)..sup.72, 77 The
primers for exon 5 produced a 160 bp product after polymerase chain
reaction (PCR) amplification which was also examined by direct
solid phase sequence analysis..sup.72, 77 The upper primer was
synthesized with a 5' biotin label and, following amplification,
the PCR product was captured by a streptavidin-linked paramagnetic
bead (Dynal) and characterized by Sanger sequencing as described
elsewhere..sup.77 Sequence polymorphisms were distinguished from
artifact by repeated analyses.
SSCP Analysis
SSCP, a method for detection of polymorphisms on the basis of
changes in migration of single-stranded DNA exposed to an electric
field,.sup.72 was carried out as set forth in Schwengel et al.,
(1994) at room temperature with and without 10% glycerol using 6%
polyacrylamide gel electrophoresis at a cross-linking monomer
concentration of 2.67%..sup.77 Four .mu.l of PCR product was mixed
with 5 .mu.l 2.times.stop buffer (95% formamide, 20 mM EDTA, 0.05%
BPB, 0.05% xylene cyanol), and 1 .mu.l 0.5% SDS and 50 .mu.M EDTA,
denatured at 85-90.degree. C. for 8 minutes, and then immediately
placed on ice. Electrophoresis was carried out at 12 watts for
approximately 24 hours for glycerol containing gels and 12 hours
for non-glycerol gels. The gels were then dried and exposed to
Kodak XAR.RTM. film.
DNA Sequencing
Direct DNA sequencing of the PCR products was accomplished using
solid phase techniques after verifying the presence of the correct
size PCR product on a 1% agarose gel stained with ethidium bromide
as set forth in Schwengel et al., (1994)..sup.77 Twenty .mu.l of
PCR product was incubated with 40 .mu.l of Dynabeads.RTM. m-280
(Dynal) for 15 minutes. The beads were washed and diluted as
suggested by the manufacturer. Each sample was subsequently washed
with B&W buffer containing 10 mM tris-HCl pH 7.5, 1 mM EDTA, 2
M NaCl, denatured with 0.1 N NaOH, and then washed with 0.1 N NaOH,
B&W buffer, and 10 mM Tris-HCl pH 8 and 1 mM EDTA (TE). The
pellet of beads was resuspended with 10 .mu.l of H2O.
Sanger sequencing reactions were carried out using Sequenase
(United States Biochemical Co.). .sup.35 S-dATP or .sup.33 P-dATP
was incorporated into the sequencing reactions, and the products
were electrophoresed through either 5% or 6% polyacrylamide gels
containing 7 M urea. Gels were dried without fixing and exposed to
X-ray film. Alleles were determined by comparing the genotypes of
parents and offspring. Infrequent artifacts were easily
distinguished from true sequence polymorphisms by repetition.
DNA was available and extracted from peripheral leukocytes. Genomic
DNA was diluted to a concentration of 200 .mu.g/ml for
amplification..sup.27, 42 Simple sequence repeats (SSR) including
DXYS154 were selected from the Genome Data Base (GDB; Welch
library, Johns Hopkins University, Baltimore, Md.). Genotyping of
the sKK-1 marker was carried out using the following primers sKK-1U
(5' CAA ATC TGA AGA GCA AAC TAT 3') (SEQ ID NO: 42) and sKK-1L [(5'
TTA AAA AAT TCA TTT CAG TAT TCT 3') (SEQ ID NO: 43) which produce a
90 bp product. Each SSR product was amplified by PCR.sup.72 and
sized according to methods previously described..sup.27, 42 Sample
handling was carried out as described by Weber et al. with minor
modifications..sup.71, 27, 42 Genotypes were determined from two
independent readings of each autoradiograph. Individuals genotyping
the families were blinded to the clinical data.
RFLP Analysis
As a result of the C to T polymorphism at position 3365, a StyI
restriction fragment length polymorphism (RFLP) was produced at
position 52 of the IL-9 exon 5 PCR product. To test for the
presence of this DNA sequence variant the lower primer from exon 5
was end-labeled prior to PCR amplification. The PCR product was
then digested with StyI producing two fragments 108 bp (labeled)
and 52 bp (unlabeled) in length. This RFLP was used along with SSCP
to confirm the presence of this polymorphism in families and
individuals.
Linkage Analyses and Data Management
Linkage analyses were performed using affected sib-pair methods
(SIBPAL, S.A.G.E.),.sup.78 an established approach for the
investigation of the genetic basis of complex traits, such as BHR,
atopy, and asthma. Affected sib-pairs are usually tested first,
since a proportion of unaffected sib-pairs may still be gene
carriers but do not express the trait. In contrast to LOD score
methods where the model of inheritance (dominant, recessive, etc.)
must be specified exactly, analysis by sib-pair methods makes no
explicit assumptions in this regard. Thus, in sib-pair analyses the
parents' clinical information is not used in testing for linkage.
The pertinent observation in these methods is how often two
affected offspring share copies of the same parental marker
allele..sup.44 If the same copy of a parental marker allele is
observed in different offspring, they are said to be inherited
"identical by descent." Linkage is suggested when affected
sib-pairs are identical by descent for a marker allele
significantly more often than expected by chance (50%). When the
same marker allele is transmitted with the disease gene in
different offspring, this implies that the marker locus is linked,
or must be located close enough on the same chromosome, to the
disease gene so they cosegregate during meiosis. The trait is then
mapped by knowing the chromosomal localization of the marker.
Linkage in humans may also be established by the method of
likelihood ratios. This method involves comparison of the
probability that observed family data would arise under one
hypothesis, for instance, linkage between two DNA markers, to the
probability that it would arise under an alternative hypothesis,
typically, nonlinkage. The ratio of these probabilities is called
the odds ratio for one hypothesis relative to the other. By
convention, mammalian geneticists prefer the log of the odds
ration, or the LOD score. Generally, linkage is considered proved
when the odds in favor of linkage versus nonlinkage become
overwhelming, or reach 1,000:1 (LOD=3). Linkage is rejected when
the odds drop to 100:1 against this hypothesis (LOD=-2). The
maximum likelihood estimate is the recombination fraction where the
likelihood ratio is largest. LODs from multiple pedigrees are thus
added until the score grows to 3 (signifying 1,000:1 odds) or falls
to -2 (indicating 100:1 odds).
All clinical and genotype data is managed using EXCELL.RTM. on a
MacIntosh.RTM. or Sun Microsystems.RTM. computer. Statistical
analyses were preformed using JMP (SAS Institute, Inc. Cary, N.C.).
The Wilcoxon/Kruskal-Wallis Tests (rank sums) was used to test
whether individuals who were homozygous (Met/Met), heterozygous
(Met/Thr), or homozygous (Thr/Thr) at codon 117 differ in their
serum total IgE. All P-values are two-tailed except affected
sib-pair analyses, where a one-tailed test was used because only an
increased sharing of alleles was expected.
Having provided this background information, applicant now
describes preferred aspects of the invention.
EXAMPLE 1
Linkage Analysis Between BHR and Murine Chromosome 13
As an aid in dissecting the complex genetic determinants of BHR,
applicant has developed murine models that differ in their genetic
susceptibility to various bronchoconstrictor stimuli. Inbred animal
models using recombinant inbred strains (BXD) can facilitate
ongoing studies in humans to determine the number of genes
regulating susceptibility to BHR, the magnitude of their affect,
and their precise chromosomal location. In particular, localizing
in an animal model a gene determining susceptibility to a critical
risk factor for asthma may aid in the positional cloning of this
gene in humans.
Although the gene(s) predisposing to BHR and atopy had not yet been
identified prior to this invention, chromosome 5q31-q33 was known
to be syntenic with portions of mouse chromosomes 11, 13, and 18.
FIG. 2 illustrates the syntenic regions containing numerous
positional candidates that may potentially play a role in airway
inflammation associated with BHR, atopy, and asthma. Specifically,
the region of human chromosome 5q31-q33 demonstrating significant
evidence for linkage with BHR is homologous to portions of mouse
chromosomes 11, 13, and 18 which contain numerous candidate
genes..sup.84
In particular, IL-9 or a nearby gene have recently been suggested
as likely candidates on the basis of linkage disequilibrium between
log serum total IgE levels and a marker in this gene using a
randomly ascertained population..sup.43
Despite comparisons with four candidate intervals, evidence for
linkage was found for only one region, designated Aib 1 (atracurium
induced bronchoconstriction 1). FIG. 3 provides the results.
Specifically, FIG. 3 sets forth the LOD score curve on mouse
chromosome 13 for atracurium-induced airway responsiveness in 24
BXD RI strains which are derived from the hyporesponsive C57BL/6J
and the hyperresponsive DBA/2J progenitor strains (solid line). The
LOD score curve resulting from the selective genotyping of 20 BXD
strains is also shown (dashed line). BXD strains -2, -6, -18, and
-32 were not used in the second analysis since they were
intermediate in phenotype displaying a mean response greater than 1
standard deviation below the DBA/2 and above the C57BL/6 mean
responses. The bronchoconstrictor response to atracurium, 20 mg/kg
given intravenously, was assessed by the change in peak inspiratory
pressure integrated over time (4 min), termed the airway pressure
time index (APTI). Atracurium-induced APTI was measured in 2-8
animals per RI strain. Marker data were obtained from the RWE data
base in the Map Manager data analysis program. The genetic distance
(cM) between markers is indicated on the abscissa. LOD scores were
calculated by the MAPMAKER/QTL linkage program. A QTL was detected
in this region and termed atracurium-induced bronchoconstriction-1
(Aib1).
FIG. 3 indicates that this quantitative trait locus (QTL) is
located on the midportion of murine chromosome 13 and attained this
interval a maximum likelihood log of the odds (LOD) of 2.42.
Forty-four percent of the total variance in atracurium-induced
bronchoconstriction was explained at Aib1 when all of the markers
in the BXD map were analyzed. The LOD for chromosome 13 increased
to 2.85 when QTL analyses were run after excluding the four strains
(BXD-2, -6, -18, and -32) that were intermediate responders to
atracurium. The known positional candidates in the linked region of
chromosome 13 include: D1 dopamine receptor (Drd1), fibroblast
growth factor receptor 4 (Fgfr4), lymphocyte antigen-28 (Ly28),
thiopurine methyltransferase (Tpmt), and IL-9.
Because the applicant was specifically testing for linkage to four
candidate regions in the mouse, based on previous mapping data in
the human, the data presented here are highly significant. As
stated in the classic paper by Lander and Botstein,.sup.67 a false
positive rate for linkage will result if the LOD threshold (T) is
chosen so that T=1/2(log10 e)(Z .alpha./n)2 (where n is equal to
the number of intervals tested). Typically a minimum LOD of 3.3 is
required as evidence of linkage..sup.67 However, this threshold is
based on the assumption that one is searching the entire genome.
These same authors point out that a LOD of 0.83 is sufficient when
only one region is examined. In this case, with four candidate
regions, a P value (.alpha.) of 0.0125 for each region is required
to obtain a true P.ltoreq.0.05, when one corrects for multiple
independent comparisons. Adopting a 5% error rate that even a
single false positive finding will occur, as suggested by Soller
and Brody,.sup.68 and solving the equation 1/2(log10 e)(Z
.alpha./n).sup.2, yields a LOD threshold of at least 1.36. A
maximal LOD of 1.48 was obtained for the Il-9 gene candidate.
Restricting the acceptable false positive error rate to
.ltoreq.0.1%, increases the LOD threshold to 2.36. Thus, the
maximal LOD generated of 2.42 for the candidate interval on
chromosome 1:3 (Aib1) is highly significant.
These LOD threshold data provide evidence of a conserved linkage
for BHR in humans and mice. BHR in humans links to the region on
chromosome 5q containing a number of growth factors and cytokines
including the IL-9 gene and the Aib1 locus maps to the IL-9 region
of murine chromosome 13.
EXAMPLE 2
Identification of an IL-9 Gene Polymorphism
Inventors demonstrated conserved linkage between the mouse and
humans for BHR. These data suggest that variation in the functions
of this gene or DNA sequence may be important in regulating
bronchial responsiveness in the mouse. Using the methods described
above, a unique product of the correct size was identified by gel
electrophoresis for each of the exons of human IL-9 after PCR. A
single polymorphism was identified by SSCP in exon 5 of the human
IL-9 gene. Direct DNA sequence analysis demonstrated a C to T
nucleotide substitution at position 3365 (GenBank accession number
M30136) of the human IL-9 gene as the cause of the novel SSCP
conformer. This DNA sequence change predicts a nonconservative
substitution of a methionine (hydrophobic) for a threonine
(hydrophilic) at amino acid 117 of the IL-9 protein.
Exon 5 codes for this segment of the protein which is within the
most highly conserved interval of human IL-9 as compared to the
mouse IL-9 sequence (see FIG. 4).
Individuals were genotyped from various populations to examine the
frequency of these alleles by direct analyses of the nucleotide
substitution in the coding sequence of human IL-9. Two of 394
individuals from a group of asthmatic families were homozygous
(Met/Met) at codon 117 (0.5%). There were 91 (23.1%) heterozygous,
and 301 (76.4%) homozygous (Thr/Thr) individuals. The true
prevalence for this IL-9 variant is likely to be significantly
higher because the Italian population of families was ascertained
through symptomatic patients with asthma. From a separate
ethnically diverse population ascertained randomly with respect to
atopy and asthma, there were 1 of 49 individuals homozygous for
(Met/Met) at codon 117 (2.0%). There were 11 (22.4%) heterozygous,
and 37 (75.5%) homozygous (Thr/Thr) individuals. The prevalence of
the Met/Thr heterozygotes was 18.9% in a fourth population
ascertained randomly with respect to atopy and asthma. Thus,
approximately 20% of the population are likely to represent
carriers of the T allele at position 3,365 as compared to the
reported sequence (GenBank accession number M30136). Because it is
well known in the art that the frequency of any allele in the
population is p2+2pq+q2, then, approximately 4% of the population
is expected to be Met/Met homozygous at codon 117 of IL-9.
Overall, serum total IgE averaged 44.5 I.U. for homozygous
individuals (Met/Met), which was significantly different from those
who were homozygous wild type (Thr/Thr) (351.7I.U.), or
heterozygous (Met/Thr) (320.9I.U.). See FIG. 5. The homozygous
protected individuals (Met/Met) failed to demonstrate evidence of
atopic allergy except for a single positive skin test in one
individual. These data indicate that this novel DNA polymorphism,
when inherited in the homozygous state, is associated with
protection from atopic allergy, including lower serum total
IgE.
FIG. 6 illustrates the PCR amplification of the IL-9 simple
sequence repeat polymorphism. This marker is compared with genotype
for these individuals for the restriction fragment length
polymorphism produced by the nucleotide polymorphism at position
3,365 as compared to the reported sequence (GenBank accession
number M30136). Two families are shown. The individuals in lanes 1
and 2 are the parents (Thr/Met) of individuals in lanes 3 (Met/Thr)
and 4 (Met/Met); lanes 5 (Thr/Thr) and 6 (Met/Met) are parents for
offspring in lanes 7 (Met/Thr) and 8 (Met/Thr). The smallest allele
for the IL-9 simple sequence repeat polymorphism (the lowest band
in each figure is 248 nucleotides in length) is in complete linkage
disequilibrium with the Met117 allele (nucleotide substitution at
position 3,365 as compared to the reported sequence (GenBank
accession number M30136)) in these individuals and in all
individuals from populations tested world wide. This was true in
both the Italian and all random ascertained ethnically diverse
individuals studied, and therefore, this marker may be used
diagnostically to detect the presence of the Met117 allele. These
data are most consistent with the hypothesis that this variant is
widely distributed in populations worldwide and arose before many
of these populations separated.
EXAMPLE 3
IL-9 Receptor Expression and Ligand Binding Assay
Purified recombinant Thr IL-9, Met IL-9, and compounds potentially
resembling Met IL-9 in structure or function are radiolabelled
using the Bolton and Hunter reagent as described in Bolton A E, and
Hunter W M, Biochem J. 133:529-539(1973). This material is labeled
to high specific activity of 2,300 cpm/fmol or greater. Human K562
and MO7e cells are grown and resuspended at 30.degree. C. in 0.8 ml
of Dulbecco's modified Eagle's medium supplemented with 10%
(vol/vol) fetal bovine serum, 50 mM 2-mercaptoethanol, 0.55 mM
L-arginine, 0.24 mM L-asparagine, and 1.25 mM L-glutamine. K562 or
MO7e cells are used as is or after transfection with the IL-9
receptor gene as described below. Plasmid DNA containing the full
length IL-9 receptor is cloned into pRC/RSV plasmid (In Vitrogen,
San Diego) and purified by centrifugation through CsCl2. Plasmid
DNA (50 micrograms) is added to the cells in 0.4 cm cuvettes just
before electroporation. After a double electric pulse (750 V/74
ohms/40 microFaradays and 100 V/74 ohms/2100 microFaradays) the
cells are immediately diluted in fresh medium supplemented with
IL-9. After 24 h the cells are washed and incubated in G418 (2.5
mg/ml, GIBCO) with either no ligand, or various concentrations of
125I-labeled ligand at 20.degree. C. for 3 h. An excess of
unlabeled ligand is used in parallel experiments to estimate
nonspecific binding. The cells are then washed, filtered, and
collected for counting. Specific incorporation is calculated by
Scatchard analysis. Similar competitive assays are run using
125I-labeled Thr117 IL-9 and various amounts of putative cold
ligands to assess specific binding.
Soluble IL-9 receptor including amino acids 44 to 270 (R&D
Systems) was incubated with different forms of human recombinant
IL-9. Varying amounts of FlagMet117 and FlagThr117 (described in
Example 7) were incubated in PBS at room temperature for 30 minutes
with 0.5 .mu.gs of soluble receptor. EBC buffer (50 mM Tris pH 7.5;
0.1 M NaCl; 0.5% NP40) was added (300 .mu.l) was added along with 1
.mu.g of anti-FLAG monoclonal antibody (IBI) and incubated for 1
hour on ice. Forty microliters of protein A sepharose solution was
added to each sample and mixed for 1 hour at 4.degree. C. Samples
were centrifuged for 1 minute 11,000.times.G and pellets were
washed 4 times with 500 .mu.g of EBC. Pellets were dissolved in 26
.mu.l of 2.times.SDS buffer, boiled for 4 minutes, and
electrophoresed through an 18% SDS polyacrylamide gel. Western
blots were performed as described in Example 15 except the blots
were probed with an anti-IL-9 receptor antibody (R&D
Systems).
FIG. 25 demonstrates the binding of the IL-9 recombinant proteins
soluble IL-9 receptor. Lane 1 is molecular weight markers, lane 2
is the IL-9 FlagMet117 incubated with the receptor, lane 3 is the
IL-9 FlagThr117 incubated with the receptor. These data demonstrate
that both forms of the recombinant IL-9 protein are bound to the
soluble receptor. Moreover, these data along with those of Example
2 (where hetrozygotes do not differ in serum Ig-E from homozygous
Thr117 individuals) are consistent with the IL-9 Met117 form
representing a weak agonist.
EXAMPLE 4
IL-9 Receptor Expression and Ligand Functional Assay in K562.
C8166-45. and MO7e Cells
Recombinant Thr117 IL-9, Met117 IL-9, and compounds potentially
resembling Met IL-9 in structure or function were purified and
prepared for use in Dulbecco's modified Eagle's medium. K562,
C8166-45 or MO7e cells are used as is or after transfection with
the IL-9 receptor gene as described in Example 3. After 24 h of
deprivation from growth factors the cells are incubated without
(control) or with variable amounts of purified Thr117 IL-9, Met117
IL-9, and compounds potentially resembling Met117 IL-9 in structure
or function. Cellular proliferation is assessed by measuring acid
phosphotase activity. Briefly, quadruplicate samples of MO7e cells
are cultured in flat-bottom microtiter plates (150 or 200
microliter wells) with or without ligand for 72 to 96 hours at 37
degrees C. Acid phosphatase is measured as suggested by the
manufacturer (Clontech, Palo Alto, Calif.). All experiments are
repeated at least twice.
EXAMPLE 5
Cell Isolation and Culture
Human peripheral blood mononuclear cells {PBMC} were isolated from
healthy donors by density gradient centrifugation using endotoxin
tested Ficoll-Paque PLUS according to the manufacturer (Pharmacia
Biotech, AB Uppsala Sweden). PBMC (5.times.10.sup.6), mouse spleen
cells (5.times.10.sup.6), or 5.times.10.sup.6 MO7e cells were
cultured in 7 ml of RPMI-1640 (Bethesda Research Labs (BRL),
Bethesda, MD) supplemented to a final concentration of 10% with
either isogenic human serum or heat-inactivated FBS. Cells were
cultured for 24 hrs at 37.degree. C. either unstimulated, or
stimulated with either PMA 5 .mu.g/ml/PHA 5 .mu.g/ml, or PHA 5
.mu.g/ml/rhIL2 50 U (R&D Systems, Minneapolis, MN).
EXAMPLE 6
RNA Isolations, RT-PCR, Cloning and Sequencing of RT-PCR
Products
Total cellular RNA was extracted after 24 hours from cultured PBMC,
mouse spleen cells, and MO7e cells using RNA PCR corekit
(Perkin-Elmer Corp, Foster City, CA) according to the supplier. One
.mu.g of RNA from each source was denatured for 5 minutes at
65.degree. C. and then reverse transcribed into cDNA using a 20
.mu.l reaction mixture (RNA PCR corekit, Perkin-elmer Corp, Foster
City, Calif.) containing 50 U of MuMLV Reverse Transcriptase, 1
U/.mu.l RNAse Inhibitor, 2.5 mM oligo d(T) 16 primer, 1 mM each of
dATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCL, pH 7.0, 25 mM
MgCl2. The reaction mixture was pipetted into thermocycler tubes,
placed in a PCR thermal cycler and subjected to 1 cycle (15 minutes
at 42.degree. C., 5 minutes at 99.degree. C., and 5 minutes at
4.degree. C). A mock reverse transcription reaction was used as a
negative control.
Then this mixture was added to a second tube containing 2 mM MgCl2,
50 mM KCl, 10 mM Tris-HCl, pH 7.0, 65.5 .mu.l of DI water, 2.5 U
Amplitaq DNA polymerase, and 1 .mu.g (20 .mu.M) each of
oligonucleotides representing human cDNA IL-9 exon 1 (forward) and
exon 5 (reverse), for a final volume of 100 .mu.l. The reaction
mixture was subjected to the following PCR conditions: 120 seconds
at 98.degree. C., then 30 cycles at: 30 seconds at 94.degree. C.;
40 seconds at 55.degree. C.; 40 seconds at 72.degree. C. Finally,
the reaction mixture was cycled one time for 15 minutes at
72.degree. C. for extension.
PCR products representing hIL-9 cDNA were subjected to gel
electrophoresis through 1.5% agarose gels and visualized using
ethidium bromide staining. Products of a mock reverse transcriptase
reaction, in which H.sub.2 O was substituted for RNA, and used as
negative control amplification in all experiments.
The PCR oligonucleotide primer pairs used in these experiments to
amplify cDNA include: human interleukin 9 (hIL,-9) exon 1 forward
5'-TCT CGA GCA GGG GTG TCC AAC CTT GGC G-3' (SEQ ID NO: 1) and exon
5 reverse 5'GCA GCT GGG ATA AAT AAT ATT TCA TCT TCA T-3' (SEQ ID
NO: 2); mouse interleukin 9 (mIL-9) exon 1 forward 5'-TCT CGA GCA
GAG ATG CAG CAC CAC ATG GGG C-3' (SEQ ID NO: 3) and mouse exon 5
reverse 5'-GCA GCT GGT AAC AGT TAT GGA GGG GAG GTT T-3' (SEQ ID NO:
4); XhoI and PvuII restriction enzyme recognition sequences are
underlined in the human and mouse IL-9 primers. PCR products were
subcloned into the TA Cloning vector (Invitrogen, San Diego, CA).
Amplification of the mouse cDNA gave a 438 bp product and
amplification of the human cDNA gave a 410 bp product.
Complementary DNAs for human IL-9 and murine mIL-9 were generated
and amplified by RT-PCR using IL-9 exon 1 and 5 specific primers
containing digestion sites for XhoI and PvuII restriction
endonucleases. Amplification products for hIL-9 and mIL-9 were
isolated from 2.5% agarose gels using silica (Sambrook et al.,
(1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor
Laboratory Press,) (incorporated herein by reference in its
entirety). After recovery, the cDNA products were ligated into the
TA Cloning vector (Invitrogen Corp., San Diego, CA) and then used
to transform INV.alpha.F' competent cells, according to the
manufacturer's instructions. Plasmids containing hIL-9 and mIL-9
cDNA inserts were isolated by conventional techniques (Sambrook et
al., (1989) Molecular Cloning: A Laboratory Manual Cold Spring
Harbor Laboratory Press). After amplification the DNA sequence
including and surrounding each insert was analyzed for PCR-induced
or cloning-induced errors.
hIL-9 and mIL-9 cDNA inserts were sequenced by the dideoxy-mediated
chain termination method (Sanger et al., (1977) Proc. Natl. Acad.
Sci. USA 74,5463-5467), using the M13 (-20) forward primer (5'-GTA
AAA CGA CGG CCA GT-3') (SEQ ID NO: 17) and Sequenase.TM. (USB), and
analyzed by gel electrophoresis (Sambrook et al., (1989) Molecular
cloning: a laboratory manual Cold Spring Harbor Laboratory Press.
hIL-9 and mIL-9 cDNA inserts without cloning and/or Taq
polymerase-induced sequence errors (see translated cDNA sequences
Figures 7 and 8) were subcloned into expression vectors (see
Figures 9-12) or used to create missense mutations and deletion
mutants.
EXAMPLE 7
Cloning and Expression of IL-9 Constructs In Vitro
General Cloning Methods for Constructs
hIL-9 was subcloned into procaryotic expression vectors. The
TA2AAF1 met and thr vectors were digested by EcoRI and the 0.420 kB
fragment (containing an XhoI site at the 5' end of the hIL9 cDNA)
was cloned into the EcoRI site contained with the polylinker of
pBluescript (PBS) (Stratagene). Clones in the sense orientation to
the T3 promoter were then digested with XhoI (the fragment
contained a 5' XhoI site from the IL-9 cDNA insert from TA vectors
and a 3' XhoI site from the PBS polylinker) and inserts were
subcloned into the XhoI sites of the procaryotic expression vectors
pGEX and pFLAG.
Cloning and Expression of IL-9 Constructs in the pGEX-4T-1
Glutathione S-transferase Gene Fusion Vector
For the expression, purification, and detection of IL-9) protein,
IL-9 cDNA inserts were subcloned into the XhoI site within the
multiple cloning cassette of the 4.9 Kb pGEX-4T-1 glutathione
s-transferase gene fusion vector (Pharmacia Biotech, Piscataway,
NJ) by standard techniques. Briefly, TA clones containing intact
IL-9 cDNA sequences, and the pGEX-4T-1 vector were digested for one
hour at 37.degree. C. using XhoI and PvuII restriction endonuclease
in the presence of 1.times.React 2 buffer (New England Biolabs,
Beverly, Mass.) (total volume 50 .mu.g). Products were
electrophoresed in a 1.5% preparative agarose gel with 10 .mu.g/ml
ethidium bromide. The appropriate sized DNA band was excised, the
agarose was melted at 45.degree. C. for 10 minutes in 3 volumes of
NaI stock solution. A silica matrix solution in DI H.sub.2 O
(Geneclean II, Lajolla, CA) was added to the solution at 5 .mu.l
per 5 .mu.g of DNA and 1 .mu.l per 0.5 .mu.g of DNA above 5 .mu.g.
The slurry was incubated at 4.degree. C. and occasionally shaken
during 30 minutes. The slurry was then pelleted via
microcentrifugation, washed 3 times in low-salt buffer and
resuspended in 10 .mu.l of DI H.sub.2 O to elute the DNA from the
silica. A final microcentrifugation provided the 10 .mu.l solution
containing purified DNA.
Products were resuspended in 50 .mu.g of DI H.sub.2 O and
precipitated by the addition of 2 volumes of ethanol and 1/10
volume 3 M sodium acetate. Samples were centrifuged at RT at 14,000
rpm for 10 minutes, air dried under negative pressure and
resuspended in an appropriate volume of DI H.sub.2 O. Ligations and
transformations of DH5a bacteria (GIBCO/BRL, Gaithersburg, Md.)
with mIL-9 and hIL-9 cDNA inserts in the pGEX-4T-1 vector were
performed using standard techniques.
To confirm that the hIL-9 cDNA inserts contained in the pGEX-4T-1
vector were of the correct nucleotide sequence, plasmids containing
candidate IL-9 cDNA were sequenced via the dideoxy-mediated chain
termination method using the aforementioned mIL-9 and hIL-9
cDNA-specific oligonucleotides (exon 1 forward, exon 5 reverse
primers).
Recombinant fusion proteins were obtained from large scale
cultures. The overnight culture of transformed E. coli (50 ml) was
inoculated into fresh LB/amp broth. The culture was incubated for 4
hr at 37.degree. C. with vigorous shaking, isopropyl
.beta.-D-thiogalactopyranoside was then added to a final
concentration of 1 mM, and the culture was incubated for an
additional 1.5 hours. The cells were harvested by centrifugation at
500.times.g at 4.degree. C. and recombinant variants were purified
by making use of affinity chromatography on glutathione-sepharose
4B column (Pharmacia) for GST-fusion proteins. Some variants were
expressed as inclusion bodies and were purified from insoluble
inclusion bodies by the procedure described by Marston (1987 The
purification of eukaryotic polypeptides expressed in E. coli in
Clover D. M. ed. DNA cloning: A practical approach, IRL Press,
Oxford, 59-88). Briefly, the cells were lysed with lysozyme
followed by treatment with deoxycholic acid. Contaminating nucleic
acids were removed by treatment with DNase I. The insoluble
material was washed once with 2 M urea and finally solubilized in
lysis buffer (50 mM Tris-Cl, pH 8.0, 1 mM EDTA, 100 mM NaCl)
containing 8 M urea. The solubilized components from the inclusion
bodies were dialyzed stepwise against decreasing concentrations of
urea (starting with 8, 6, 4 and 2 M of urea) in lysis buffer to
allow for refolding of the denatured protein. Finally, the sample
was dialyzed against 2 M urea and 2.5% .beta.-mercaptoethanol
(.beta.-ME) and centrifuged at 10,000 g for 15 minutes. The fusion
protein was finally dialyzed against 0.01 M Tris-Cl, pH 8.0. Fusion
proteins expressed in pGEX-4T vector were cleaved with 100 U of
Thrombin for 6 hours at 37.degree. C. and recovered in flow through
fractions after chromatography on glutathione-Sepharose 4B column.
Final purification was achieved by chromatography on Sephadex G-100
column (100.times.1.5 cm), packed and equiliberated with 0.05 M
ammonium bicarbonate buffer.
Cloning and Expression of IL-9 Constructs in the pFLAG-1.TM.
Expression Vector
For the expression, purification and detection of human IL-9
protein, IL-9 cDNA inserts were subcloned into the Xho2 site of the
multiple cloning site (XhoI) of the 5.37 Kb Flag vector. FLAG
technology is centered on the fusion of a low molecular weight (1
kD), hydrophilic, FLAG marker peptide to the N-Terminus of a
recombinant protein expressed by the pFLAG-1.TM. Expression
Vector(1) (obtained from IBI Kodak). Each bacterial colony was
grown in LB broth containing 50 .mu.g ampicillin per ml until the
optical density at 590 nm reached O/0.6. IPTG was then added to a
final concentration of 1 mM, and the cultures incubated for an
additional 1 hr to induce protein synthesis. The cells were
harvested by centrifugation, and the cell pellet was boiled in 50
.mu.l of Laemmli buffer (Laemmli, 1970)) for 10 minutes and
electrophoresed on 10% polyacrylamide gels. The Anti-FLAG.TM. M1
monoclonal antibody was used for specific and efficient detection
of the FLAG fusion protein on western (see FIG. 13) slot or dot
blots throughout its expression, affinity purification, and FLAG
marker removal. The FLAG fusion protein was rapidly purified under
mild, non-denaturing conditions in a single step by affinity
chromatography with the murine Anti-FLAG.TM. IgG M1 monoclonal
antibody covalently attached to agarose. Following affinity
purification the fusion protein may be used after removal from the
affinity column or the authentic protein may be recovered in
biologically active form by specific and efficient proteolytic
removal of the FLAG peptide with enterokinase. Final purification
was achieved by chromatography on Sephadex G-100 column
(100.times.1.5 cm), packed and equiliberated with 0.05 M ammonium
bicarbonate buffer. The promoters described above in this example
may also be used with FLAG technology.
SDS-PAGE and Immunoblot Analysis
SDS-PAGE was performed by the method of Laemmli (Laemmli U.K.
(1970) Nature 227, 680-685)(incorporated herein by reference in its
entirety) by using a 12.5% polyacrylamide gel in a mini-gel system
(SE 280 vertical gel unit, Hoefer). For immunoblot analysis, the
proteins separated by SDS-PAGE were transferred to nitrocellulose
membranes by using the TE 22 Mighty small transfer unit (Hoefer) in
25 mM Tris-glycine buffer, pH 8.3, containing 15% methanol (Towbin
et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354). The
unoccupied binding sites on the membrane were blocked by incubating
for 1 h with 20 mM Tris-HCl buffer, pH 8.0, containing 2% bovine
serum albumin. The membranes were then incubated with 1:200
dilution of antibodies overnight at 4.degree. C. The membranes were
washed and treated with 1:2000 diluted goat anti-rabbit IgG
conjugated with either peroxidase or alkaline phosphotase for 1 h.
After washing, the bound antibodies were visualized by addition of
the super-substrate chemiluminescent reagent (Pierce) or the
4-chloro-1-naphthol color developing reagent. The reaction was
stopped by immersing the membranes in distilled water. FIG. 13
demonstrates that the purified recombinant human FLAG IL-9 fusion
proteins (Met117 and Thr117) are the correct size and in the
correct reading frame because they are recognized by the
Anti-FLAG.TM. M1 monoclonal antibody.
Analytical Methods
The molecular mass of the purified proteins was confirmed by matrix
assisted laser desorption mass spectrometry using Perceptive
Biosystems Voyager Biospectrometry workstation. Amino acid analyses
were performed after hydrolysis of the sample in 6N HCl at 110-C.
for 24 h in evacuated sealed glass bulbs.
Automated Edman Degradation
The partial amino acid sequence of the purified proteins is
determined by automated step-wise sequencing on an Applied
Biosystems model 477A gas-phase sequencer with an on-line model 20A
PTH analyzer.
EXAMPLE 8
Deletion of Exon 2 and/or Exon 3: Mutagenesis and Sequencing of
Constructs
Human exon 2 and exon 3 deletions are created using ExSite
PCR-based site-directed mutagenesis kit as suggested by the
manufacturer (Stratagene, La Jolla, Calif.). The PCR primers are as
follows:
h9CD1U forward 5'-GTG ACC AGT TGT CTC TGT TTG-3' (SEQ ID NO: 5);
h9CD1L reverse 5'-CTG CAT CTT GTT GAT GAG GAA-3' (SEQ ID NO: 6);
h9CD2U forward 5'-GAC AAC TGC ACC AGA CCA TGC-3' (SEQ ID NO: 7);
h9CD2L reverse 5'-ATT AGC ACT GCA GTG GCA CTT-3' (SEQ ID NO:
8).
Exon 2 deletions are created by using the primer pair h9CD1L
forward and h9CD1L reverse. Exon 3 deletions are created by using
h9CD2U forward and h9CD2L reverse. Deletions that included exon 2
and exon 3 use the primer pair h9CD2U forward h9CD1L reverse.
Mouse exon 2 and exon 3 deletions are created using ExSite
PCR-based site-directed mutagenesis kit as suggested by the
manufacturer (Stratagene, La Jolla, Calif.). The PCR primers are as
follows: m9CD1U forward
m9CD1U forward 5'-GTG ACC AGC TGC TTG TGT CTC-3' (SEQ ID NO: 9);
m9CD1L reverse 5'-CTT CAG ATT TTC AAT AAG GTA-3' (SEQ ID NO: 10);
m9CD2U forward 5'-GAT GAT TGT ACC ACA CCG TGC-3' (SEQ ID NO: 11);
m9CD2L reverse 5'-GTT GCC GCT GCA GCT ACA TTT-3' (SEQ ID NO:
12).
Exon 2 deletions are created by using the primer pair m9CD1U
forward and m9CD1L reverse. Exon 3 deletions are created by using
m9CD2U forward and m9CD2L reverse. Deletions that included exon 2
and exon 3 use the primer pair m9CD2U forward m9CD1L reverse.
Mutagenized constructs of the hIL-9 and mIL-9 cDNA inserts are
sequenced by the dideoxy-mediated chain termination method (Sanger
et al. (1977) Proc. Natl. Acad. Sci. USA 74,5463-5467)
(incorporated herein by reference in its entirety), using the M13
(-20) forward primer (5'-GTAAAACGACGGCCAGT-3') (SEQ ID NO: 18) and
Sequenase.TM.(USB), with analysis by gel electrophoresis (Sambrook,
J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press). Mutants that lack exon 2, exon 3,
or both exon 2 and exon 3, and are without Taq polymerase-induced
sequence errors can be used to create expression vectors.
EXAMPLE 9
Cell Lines, Cellular Proliferation Assays and Inhibition of IL-9
Activity
Cell lines were used to assess the function of peptides,
aminosterols, tyrophostins, rhIL-9, rmIL-9, and recombinant mutant
forms of these proteins as well as all other compounds that block
IL-9 function. A proliferative response was measured and compared
to each of the other cytokines, variant or mutant forms of Il-9, or
IL-9 antagonists. In addition, compounds were tested for their
ability to antagonize the baseline proliferative response. Once a
baseline proliferative response was established for a cytokine a
statistically significant loss of response in assays repeated three
times in triplicate was considered evidence for antagonism. A true
antagonistic response was differentiated from cellular toxicity by
direct observation, trypan blue staining (a technique well known to
one of normal skill in the art), and loss of acid phosphatase
activity. Specificity was assessed for the antagonist by evaluating
whether the activity was substantially expressed against other
proliferative agents such as steel factor, interleukin 3, or
interleukin 4.
The MO7e line is a human megakaryoblastic cell line, cultured in
RPMI 1640 (GIBCO/BRL, Gaithersburg, Md.), 20% Fetal Bovine Serum
(Hyclone) and 10 ng/ml IL-3 (R&D Systems, Minneapolis, Minn.).
The MJ line is a cytokine independent human lymphoblastoid cell
line grown in RPMI 1640 (GIBCO/BRL) K562 is a human erthroleukemia
cell line, cultured in RPMI 1640 (GIBCO/BRL) and 10% fetal bovine
serum (Hyclone). C8166-45 is a IL-9 receptor bearing line, cultured
in RPMI 1640 GIBCO/BRL) and 10% Fetal bovine serum (Hyclone). All
the cell lines respond to cytokines including IL-9. The cell lines
are fed and reseeded at 2.times.10.sup.5 cells/ml every 72
hours.
The cells were centrifuged for 10 minutes at 2000 rpm and
resuspended in RPMI 1640 with 0.5% Bovine Serum Albumin (GIBCO/BRL,
Gaithersburg, Md.) and insulin-transferrin -selenium (ITS)
cofactors (GIBCO/BRL, Gaithersburg, Md.). Cells were counted using
a hemocytometer and diluted to a concentration of 1.times.10.sup.5
cells/ml and plated in a 96-well microtiter plate. Each well
contained 0.15 or 0.2 ml giving a final concentration of
2.times.10.sup.4 cells per well.
MO7e cells were stimulated with 50 ng/ml Stem Cell Factor (SCF)
(R&D Systems, Minneapolis, MN) alone, 50 ng/ml SCF plus 50
ng/ml IL-3 (R&D Systems, Minneapolis, MN), or 50 ng/ml SCF plus
50 ng/ml IL-9. A control was included which contains cells and
basal media only. Serial dilutions of test compounds (i.e,
recombinant IL-9 proteins, peptides, small molecules) were added to
each test condition in triplicate. The MJ cell line was used as an
independent control for nonspecific cytotoxicity. Cultures were
incubated for 72-96 hours at 37.degree. C. in 5% CO.sub.2.
Cell proliferation was assayed using the Abacus Cell Proliferation
Kit (Clontech, Palo Alto, CA) which determines the amount of
intracellular acid phosphatase present as an indication of cell
number. The substrate p-nitrophenyl phosphate (pNPP) was converted
by acid phosphatase to p-nitrophenol which was measured as an
indicator of enzyme concentration. pNPP was added to each well and
incubated at 37.degree. C. for one hour. 1N sodium hydroxide was
then added to stop the enzymatic reaction, and the amount of
p-nitrophenol was quantified using a Dynatech 2000 plate reader
(Dynatech Laboratories, Chantilly, VA) at 410 nm wavelength.
Standard curves that compare cell number with optical absorbance
were used to determine the linear range of the assay. Assay results
were only used when absorbance measurements are within the linear
range of the assay.
FIG. 14 illustrates the amino acid sequence of three peptide
antagonists of IL-9 function. Each peptide was incubated with MO7e
cells and inhibition of cellular growth induced by IL-9 was
determined by comparison with control conditions (no peptide) (see
Figures 15-17). There was no evidence for cytotoxicity with any of
the peptides. Peptides KP-16 and KP-20 are predicted to lie within
two anti-parallel alpha-helicies and define a critical IL-9
receptor binding domain for the IL-9 ligand. The protein
polymorphism at codon 117 lies within KP-23 and KP-24 which also
exhibited antagonistic properties, further demonstrating the
importance of this region surrounding the site of genetic
variation.
FIG. 18 illustrates the effect of tyrophostins (obtained from
Calbiochem) on the IL-9 dependent growth of MO7e cell in vitro.
Each tyrophostin was incubated with MO7e cells and inhibition of
cellular growth induced by IL-9 was determined by comparison with
control conditions (no treatment). There was no evidence for
cytotoxicity with any of the treatments. Tyrophostins B46 and B56
provided the greatest inhibition suggesting a common structure
activity relationship.
FIG. 19 illustrates the effect of aminosterols isolated from the
shark liver as set forth in U.S. patent application Nos. 5,637,691;
5,733,899; 5,721,226; and/or 5,840,740, incorporated herein by
reference on the IL-9 dependent growth of MO7e cell in vitro. Each
aminosterol was incubated with MO7e cells at 20 .mu.g/ml of the
culture media and inhibition of cellular growth induced by IL-9 was
determined by comparison with control conditions (no treatment).
There was no evidence for cytotoxicity with any of the treatments.
Aminosterols 3 and 6 consistently provided the greatest inhibition
of growth.
EXAMPLE 10
Assay for Proliferation of IgE Secreting Cells
B cell lines can be used to assess the function of rIL-9 and
recombinant mutant forms of these proteins as well as other IL-9
antagonists. The proliferation of IgE secreting cells is measured
for rIL-9 and compared to other cytokines or variant forms of
rIL-9. In addition, compounds are tested for their ability to
antagonize the baseline proliferative response of IgE secreting
cells to rIL-9. Once a baseline IgE response is established for a
cytokine, a statistically significant (P<0.05) loss of response
in assays repeated three times in triplicate is considered evidence
for antagonism. A true antagonistic response is differentiated from
cellular toxicity by trypan blue staining (a technique well known
to one of normal skill in the art).
Cell Preparation and Cultures
Peripheral blood lymphocytes (PBL) are isolated from heparinized
blood of healthy donors or by mincing the spleens of mice.
Mononuclear cells are separated by centrifugation on Ficoll/Hypaque
(Pharmacia, Uppsala, Sweden) gradients. Semi-purified human B
lymphocytes are obtained by resetting with neuraminidase (Behring,
Marburg, FRG)--treated sheep red blood cells and plastic adherence
for 1 hour at 37.degree. C. B cells are also purified using
paramagnetic separation with anti-CD20 coated magnetic beads
(DYNAL) according to the manufacturer's recommendations.
The relative proportion of B cells, T cells and monocytes is
determined by flow cytometry using monoclonal antibodies specific
for CD23, CD3 and CD14, respectively (Becton Dickinson, Mountain
View, Calif.). Briefly, 10.sup.6 cells/ml are incubated with a
1:1000 dilution of phycoerythrin conjugated anti-CD23 and
fluorescein-conjugated anti-CD3 and anti-CD14 antibodies for 30
minutes at 4.degree. C. After 3 spin washes with sterile PBS and 1%
bovine serum albumin (Sigma) fluorescence is measured with a
cytofluorograph (FACSTAR Plus, Becton Dickinson, Grenoble, France).
Typically, there are 45% CD20+, 35% CD3+ and 10% CD14+ cells in a
count of 5000 cells per sample.
Cells are cultured at a density of 2.times.10.sup.6 cells/ml RPM1
1640 supplemented with 10% heat-inactivated fetal calf serum (FCS),
2 mM glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin and
20 mM HEPES (RPM1-FCS) at 37.degree. C. under a 5% CO2/95% air
humidified atmosphere. Cultures are incubated with increasing
concentrations of IL-4, rhIL-9, rmIL-9, or recombinant mutant forms
of these proteins, alone, or in combination. Competition
experiments are run with mixtures of one or more of these
recombinant molecules or other IL-9 antagonists. The cultures are
maintained for 9-13 days.
Frequency of IgE-secreting B Cells
The frequency of IgE-secreting human B cells in response to human
or murine IL-9 is determined using an ELISA-spot assay (Dugas et
al., (1993) Eur J Immunol 23, 1687-1692; Renz, H. et al., (1990) J.
Immunology. 145, 3641-3644. Nitrocellulose flat-bottomed 96-well
plates are coated overnight at 4.degree. C. with purified goat
antihuman IgE mAb diluted in 0.1 M NaHCO3 buffer (2.5 .mu.g/ml).
After one PBS-Tween 20 wash, plates are incubated for 1 hour with
RPMI-FCS to saturate nonspecific binding sites. B cells obtained
after 9-13 days of culture are collected, washed thrice and
resuspended at 10.sup.5 cells/ml RPMI-FCS, then transferred onto
the anti-IgE-coated plates followed by an 18 hour incubation at
37.degree. C. A peroxidase-conjugated mouse antihuman IgE mab at
various dilutions is added for 2 hours at 37.degree. C. after
washing. Spots are visualized after addition of diamino-benzidine
diluted in 0.1 M Tris-HCl containing 0.03% H.sub.2 O.sub.2. After
24 hours spots are counted with an inverted microscope at
25.times.magnification. Data are expressed as the number of
IgE-secreting cells per 10.sup.6 cells.
EXAMPLE 11
ELISA for IgE Secreted by Cells Co-stimulated With IL-9
Cells are isolated, prepared and stimulated as described above in
Example 10. Flat bottom microtiter plates (Nunc) are coated with
rabbit anti-human IgE (1:2000, final dilution; Serotec, Oxford,
GB), in 200 .mu.l of 10 mM bicarbonate buffer (pH 9.6). After
overnight incubation at 40.degree. C., the plates are washed four
times with phosphate-buffered saline (PBS) containing 0.05% Tween
(PBS-Tween; Merck, Hohenbrunn, FRG) and are incubated for 1 h at
room temperature with RPMI-FCS to saturate nonspecific
protein-binding sites. After washing, 200 .mu.l serial dilutions of
human IgE (Eurobio, Les Ulis, France), standards in PBS-Tween are
added to the respective plates to establish calibration curves.
Dilutions of culture supernatants to be tested are then added and,
after 2 h at room temperature, the plates are washed and 200 .mu.l
of diluted specific alkaline phosphatase-conjugated anti-IgE
(1:250; Serotec), anti-IgG or anti-IgM (Behring) is added in the
appropriate plates. After 2 h at room temperature, the plates are
washed and 200 .mu.l (0.5 mg/ml) p-nitrophenylphosphate (Sigma) in
citrate buffer is added. Plates are incubated at 37.degree. C., and
absorbance (A) is measured at 405 nm using an autoreader (Dynatech
Laboratories Inc, Chantilly, Va.). The threshold sensitivities of
the assays are 100 pg/ml for IgE, 1 ng/ml for IgG, and 2 ng/ml for
IgM and the variation between duplicate determinations of samples
typically does not exceed 10%.
EXAMPLE 12
The Role of IL-9 in Murine Models of Asthma: The Airway Response of
Unsensitized Animals
Animals
Certified virus-free male mice ranging in age from 5 to 6 weeks
were obtained from the Jackson Laboratory (Bar Harbor, ME). Animals
were housed in high-efficiency particulate filtered air (HEPA)
laminar flow hoods in a virus and antigen free facility and allowed
free access to pelleted rodent chow and water for 3 to 7 days prior
to experimental manipulation. The animal facilities were maintained
at 22.degree. C. and the light:dark cycle was automatically
controlled (10:14 h light:dark). Male and female DBA/2 (D2),
C57BL/6 (B6), and (B6D2)F1 (F1) mice 5 to 6 weeks of age were
purchased from the Jackson Laboratory, Bar Harbor, ME, or the
National Cancer Institute, Frederick, Md. BXD mice were purchased
from the Jackson Laboratory, Bar Harbor, Me. Food and water were
present ad libitum.
Phenotyping and Efficacy of Pretreatment
To determine the bronchoconstrictor response, respiratory system
pressure was measured at the trachea and recorded before and during
exposure to the drug. Mice were anesthetized and instrumented as
previously described. (Levitt et al., (1988) FASEB J. 2, 2605-2608
(1988); Levitt et al., (1989), J. Appl. Physiol. 67, 1125-1132;
Kleeberger et al., (1990) Am. J. Physiol. 258, L313-320; Levitt
(1991) Pharmacogenetics 1, 94-97; Levitt et al., (1995) Am. J.
Respir. Crit. Care Med. 151, 1537-1542; Ewart et al., (1995) J.
Appl. Phys. In Press. Airway responsiveness was measured to one or
more of the following: 5-hydroxytryptamine (5HT) (sigma). An
additional branch construction that can be used is acetylcholine
(sigma), atracurium (Glaxo welcome). A simple and repeatable
measure of the change in Ppi following bronchoconstrictor challenge
was used and which has been termed the Airway Pressure Time Index
(APTI) (Levitt et al., (1988) FASEB J. 2, 2605-2608; Levitt et al.,
(1989) J. Appl. Physiol . 67, 1125-1132. The APTI was assessed by
the change in peak inspiratory pressure (Ppi) integrated from the
time of injection till the peak pressure returned to baseline or
plateaued. The APTI was comparable to airway resistance (Rrs),
however, the APTI includes an additional component related to the
recovery from bronchoconstriction.
The strain distribution of bronchial responsiveness was identified
in multiple inbred mouse strains in previous studies (Levit et al.,
(1988) FASEB J 2, 2605-2608; Levitt et al., (1989) J. Appl. Physiol
. 67, 1125-1132. The Rrs and/or APTI was determined in A/J,
C3H/HeJ, DBA/2J, C57BL/6J mice.
Prior to sacrifice whole blood was collected for serum IgE
measurements by needle puncture of the inferior vena cava in
completely anesthetized animals. The samples were spun to separate
cells and serum was collected and used to measure total IgE levels.
Samples not measured immediately were frozen at -20.degree. C.
Bronchoalveolar lavage and cellular analyses was preformed as
described elsewhere (Kleeberger et al., (1990)).
All IgE serum samples were measured using an ELISA
antibody-sandwich assay. Microtiter plates (Corning #2585096,
Corning, NY) were coated, 50 .mu.l per well, with rat anti-mouse
IgE antibody (Southern Biotechnology #1130-01, Birmingham, AL) at a
concentration of 2.5 .mu.g/ml in coating buffer of sodium
carbonate-sodium bicarbonate with sodium azide (Sigma #S-7795,
#S-6014 and #S-8032, St Louis, MO). Plates were covered with
plastic wrap and incubated at 4.degree. C. for 16 hours. The plates
were washed three times with a wash buffer of 0.05% Tween-20 (Sigma
#P-7949) in phosphate-buffered saline (BioFluids #313, Rockville,
Md.), incubating for five minutes for each wash. Blocking of
nonspecific binding sites was accomplished by adding 200 .mu.l per
well 5% bovine serum albumin (Sigma #A-7888) in PBS, covering with
plastic wrap and incubating for 2 hours at: 37.degree. C. After
washing three times with wash buffer, duplicate 50 .mu.l test
samples were added to the wells. Test samples were assayed after
being diluted 1:10, 1:50, and 1:100 with 5% BSA in wash buffer. In
addition to the test samples a set of IgE standards (PharMingen
#03121D, San Diego, CA) at concentrations from 0.8 ng/ml to 200
ng/ml in 5% BSA in wash buffer were assayed to generate a standard
curve. A blank of no sample or standard was used to zero the plate
reader (background). After adding samples and standards, the plate
was covered with plastic wrap and incubated for 2 hours at room
temperature. After washing three times with wash buffer, 50 ul of
second antibody rat anti-mouse IgE-horseradish peroxidase conjugate
(PharMingen #02137E) was added at a concentration of 250 ng/ml in
5% BSA in wash buffer. The plate was covered with plastic wrap and
incubated 2 hours at room temperature. After washing three times
with wash buffer, 100 ul of the substrate 0.5 mg/ml
0-phenylaminediamine (Sigma #P-1526) in 0.1 M citrate buffer (Sigma
#C-8532) was added to every well. After 5-10 minutes the reaction
was stopped with 50 .mu.l of 12.5% H.sub.2 SO.sub.4 (VWR #3370-4,
Bridgeport, N.J.) and absorbance was measured at 490 nm on a
Dynatech MR-5000 plate reader (Chantilly, VA). A standard curve was
constructed from the standard IgE concentrations with antigen
concentration on the x axis (log scale) and absorbance on the
y-axis (linear scale). The concentration of IgE in the samples was
interpolated from the standard curve.
EXAMPLE 13
The Role of IL-9 in Murine Models of Asthma: The Airway Response of
Sensitized Animals
Animals, Phenotyping, and Optimization of Antigen Sensitization
Animals and handling were essentially as described in Example 12.
Sensitization with turkey egg albumin (OVA) and. aerosol challenge
was carried out to assess the effect on BHR, BAL, and serum IgE.
OVA was injected I.P. (25 .mu.g) day 0 prior to OVA or saline
aerosolization. Mice were challenged with OVA, or saline
aerosolization which was given once daily for 5 to 7 days starting
on either day 13 or 14. Phenotypic measurements of serum IgE, BAL,
and BHR was carried out on day 21. The effect of a 7 day OVA
aerosol exposure on bronchoconstrictor challenge with 5-HT and
acetylcholine were evaluated along with serum total IgE, BAL total
cell counts and differential cell counts, and bronchial
responsiveness. The effect of antibody (Ab) or saline pretreatment
on saline aerosol or OVA aerosol induced lung inflammation was
examined by measuring BHR, BAL, and serum IgE. Ab were administered
I.P. 2-3 days prior to aerosolization of saline or OVA.
Lung histology was carried out after the lungs are removed during
deep anesthesia. Since prior instrumentation may introduce
artifact, separate animals were used for these studies. Thus, a
small group of animals was treated in parallel exactly the same as
the cohort undergoing various pretreatments except these animals
were not used for other tests aside from bronchial responsiveness
testing. After bronchial responsiveness testing, the lungs were
removed and submersed in liquid nitrogen. Cryosectioning and
histologic examination were carried out in a routine fashion.
Polyclonal neutralizing antibodies for murine IL-9 were purchased
from R & D systems, Minneapolis, MN and blocking antibodies for
murine IL-9 receptor were produced for Magainin Pharmaceuticals
Inc. by Lampine Biological Labatories, Ottsville, Pa. using peptide
conjugates produced at Magainin. The polyclonal antisera were
prepared in rabbits against peptide sequences from the murine IL-9
receptor. The peptides used to produce the antisera were:
GGQKAGAFTC (residues 1-10)(SEQ ID NO:19); LSNSIYRIDCHWSAPELGQESR
(residues 11-32)(SEQ ID NO:20); and CESYEDKTEGEYYKSHWSEWS (residues
184-203 with a Cys residue added to the N-terminus for coupling the
peptide to the carrier protein)(SEQ ID NO:21). The antisera were
generated using techniques described in Protocols in Immunology,
Chapter 9, John Wiley & Sons. Briefly, the peptides were
coupled to the carrier protein, Keyhole Limpet, hemocyanin,(Sigma)
through the side chain of the Cys residue using the bifunctional
cross-linking agent MBS (Pierce). Peptide conjugates were used to
immunize rabbits with appropriate adjuvents and useful antisera was
obtained after several booster injections of the peptide conjugate.
The antibodies were used therapeutically to down regulate the
functions of IL-9 and assess the importance of this pathway to
baseline lung responsiveness, serum IgE, and BAL in the
unsensitized mouse. After Ab pretreatment on baseline BHR, BAL, and
serum IgE levels relative to controls was determined. In additional
experiments, recombinant human and murine IL-9 were administered
I.P. 1 day before and daily during antigen sensitization (days
13-18). The animals were then phenotypes as described.
The phenotypic response of a representative animal treated with
saline I.P. on day zero and challenged on days 14-20 with saline
(as described in Example 12) is shown in FIG. 20A panel 1 (top).
Baseline (control) serum total IgE was 9.2 ng/ml. Bronchoalveolar
lavage (BAL) total cell counts showed 182,500 cells per milliliter
of BAL. These animals did not demonstrate bronchial
hyperresponsiveness when compared to historical controls (Levitt et
al., (1989) J. Appl. Physiol . 67, 1125-1132).
FIG. 20A panel 2 (top middle) shows a representative animal from a
group presensitized with OVA I.P on day zero and challenged with
saline on days 14-20. These animals did not differ in their
response to bronchoconstrictor, serum IgE, or BAL cell counts from
the unsensitized mice (FIG. 7 top panel).
FIG. 20B panel 3 (bottom middle) shows a representative animal from
those presensitized with OVA I.P on day zero and challenged with
antigen (OVA) on days 14-20. These animals developed bronchial
hyperresponsiveness (approximately two to three-fold over
controls), elevated serum IgE (nearly one thousand-fold over
controls), and increased numbers of inflammatory cells in the
airway as demonstrated by elevated. BAL cell counts (approximately
thirty-fold) as compared to controls (FIGS. 20A and 20B top 2
panels). Most of the cells recruited to the airway as a result of
this antigen challenge were eosinophils.
FIG. 20B panel 4 (bottom) shows a representative animal from those
presensitized with OVA I.P on day zero, pretreated with polyclonal
neutralizing antibodies for murine IL-9 (approximately 200
.mu.g/mouse I.P. in 0.5 ml of PBS), and challenged with antigen
(OVA) on days 14-20. These animals were protected from the response
to antigen. They did not differ significantly in their bronchial
responsiveness, serum IgE, or BAL cell counts from controls (FIGS.
20A and 20B top 2 panels).
FIG. 21 illustrates the effect of antigen challenge to OVA (as
described above) with and without pretreatment with polyclonal
neutralizing antibodies to murine IL-9 I.P. three days prior in
representative animals. The left figure (Al-2-1B) is a histologic
section from the lungs of control animals (sensitized to OVA but
exposed only to a saline aerosol challenge). The middle figure
(Al-3-5) is a histologic section from the lungs of animals
sensitized to OVA and exposed to an OVA aerosol challenge. The
right figure (Al-4-5) is a histologic section from the lungs of
animals sensitized to OVA and exposed to an OVA aerosol challenge
who were pretreated three days prior with polyclonal neutralizing
antibodies to murine IL-9. Pretreatment with neutralizing antibody
produced histological confirmation of complete protection from
antigen challenge.
FIG. 22 panel 1 (top) shows a representative animal from mice
presensitized with OVA I.P. on day zero and challenged with antigen
(OVA) on days 13-18. These animals developed bronchial
hyperresponsiveness (approximately two to three-fold over
controls), and increased numbers of inflammatory cells including
eosinphils in the airways as demonstrated by elevated BAL cell
counts as compared to controls (FIGS. 20A and 20B top 2 panels).
Many of the cells recruited to the airway as a result of this
antigen challenge were eosinophils.
FIG. 22 panel 2 (bottom) shows a representative animal from those
presensitized with OVA I.P. on day zero, pretreated with polyclonal
neutralizing antibodies to the murine IL-9 receptor (approximately
1 mg/mouse I.P. in 0.5 ml of PBS), and challenged with antigen
(OVA) on days 13-18. This representative animal was protected from
the response to antigen. This response did not differ significantly
bronchial responsiveness, BAL cell counts from controls (FIGS. 20A
and 20B top 2 panels). These data demonstrate the potential
effectiveness of treating atopic allergy with antibodies to the
IL-9 receptor.
EXAMPLE 14
Murine Spleen Isolation and Culture
Mice were anesthetized and spleens were removed aseptically.
Spleens were minced with scissors and gently passed through a wire
mesh (autoclaved) (#60 sieve). Cells were resuspended in 40 mls of
RPMI-1640 (GIBCO, BRL, Rockville, Md.), and spun for 5 min. at
250.times.G twice. The pellet was resuspended in 10 mls of lysing
butter to remove RBCs (4.15 gm NH4Cl, 0.5 gm KHCO3; 019 g EDTA to
500 mls with ddH20]. Cells were incubated for about 5 minutes at
37.degree. C. and 40 mls of RPMI-FCS (RPMI-1640, 10% AFBS, 50 .mu.M
BME: 2 mM glutamine, containing penicillin and streptomycin). These
cells were spun again for 5 minutes at 250.times.G and resuspended
in 20 mls RPMI-FCS with or without 5 .mu.g/ml of concanavalin A
(Sigma #C5275). IL-9 was assessed at 48 hours in untreated
splenocytes and after concanavalin A stimulation from DBA/2J (D2)
and C57BL/6J (B6) mice. IL-9 was amplified by RT-PCR (as set forth
in Example 6), and probed with an IL-9 specific murine probe after
Southern transfer. Southern blots were performed by "standard"
techniques. Briefly, RT-PCR products were electrophoresed in 2%
agarose gels. Gels were stained with ethidium bromide and
photographed with a ruler to determine molecular weight of DNA in
southern blot. Gels were then soaked in 0.5N NaOH for 30 minutes
and neutralized in 0.5M Tris, pH 7.0 for 30 minutes. DNA was
transferred to zetaprobe (BioRAD) nylon membrane by capillary
transfer in 20.times.SSC overnight. The next day, the membrane was
air dried, baked at 80.degree. C. for 15 minutes and prehybridized
in 6.times.SSC and 0.1% SDS for 1 hour at 42.degree. C. A kinase
end-labelled p32 oligonucleotide probe
(5'-AATTACCTTATTGAAAATCTGAAG-3') (SEQ ID NO: 44) was added to the
hybridization solution plus 0.1 mg/ml sheared salmon sperm DNA and
incubated overnight at 42.degree. C. The next day, the filter was
washed in 3xSSC and 0.1% SDS at 37.degree. C. for 30 minutes, and
the filter was exposed to film for 1 hour. FIG. 26 illustrates
steady state levels of IL-9 after 48 hours from each strain of
mice. IL-9 was observed in unstimulated D2 (D2-) splenocytes,
whereas no IL-9 was detectable in B6 (B6-) mice. While there was a
significant increase of IL-9 after concanavalin A stimulation in D2
(D2+) splenocytes, there was no detectable It-9 in B6 (B6+) mice
despite concanavalin A treatment.
EXAMPLE 15
Expression of Human Met117 IL-9 and Thr117 IL-9 in PBMCs SDS-PAGE
and Immunoblot Analysis
After obtaining proteins isolated from human PBMC of healthy donors
inhibiting either the wild type (Thr117) or Met117-IL-9 genotypes
as set forth in Example 13, and SDS-PAGE was performed by the
method of Laemmli (Laemmli U.K. (1970) Nature 227, 680-685) by
using a 18% polyacrylamide gel in a mini-gel system (Xcell II
vertical gel unit, Novex). For immunoblot analysis, the proteins
separated by SDS-PAGE were transferred to nitrocellulose membranes
by using the SD transblot transfer unit (Biorad) in 25 mM
Tris-glycine buffer, pH 8.3, containing 15% methanol (Towbin et
al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354). The
unoccupied binding cites on the membrane were blocked by incubating
for 1 hour to overnight with 20 mM Tris-HCl buffer, pH 8.0, plus
0.05% tween 20 (TBST) containing 5% dry milk. The membranes were
then incubated with 1:1000 dilution of goat anti-human IL-9
polyclonal antibody (R&D Systems) for 1 hour at room
temperature. The membranes were washed with TBST and treated with
1:10,000 dilution of mouse anti-goat TgG conjugated with
horseradish peroxidase for 1 hour. After washing with TBST, the
bound antibodies were visualized by addition of the super signal
substrate chemiluminescence system (Pierce).
FIG. 24 demonstrates the expression of human IL-9 proteins from
cultured PBMCs 48 hours after mitogen stimulation in individuals
whose genotypes have been determined by genomic analysis of the
IL-9 gene. Lane 1 is molecular weight markers, lane 2 is a Met117
homozygote, lane 3 is a heterozygote Met117/Thr117, lane four is a
Thr117 homozygote. A single product of the approximate expected
size (14 kD) was seen in each individual PBMCs after mitogen
stimulation. These data demonstrate that both forms of the IL-9
protein are expressed and stable at steady state.
While the invention has been described and illustrated herein by
references to various specific materials, procedures and examples,
it is understood that the invention is not restricted to the
particular material combinations of material, and procedures
selected for that purpose. Numerous variations of such details can
be implied as will be appreciated by those skilled in the art.
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Other embodiments of the invention described above and will be
apparent to those skilled in the art from consideration of the
specification and practice of the invention disclosed within. It is
intended that the specification and examples considered as
exemplary only, with true scope and spirit of the invention being
indicated by the following claims:
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 44 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
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<400> SEQUENCE: 1 tctcgagcag gggtgtccaa ccttggcg 28
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
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gcagctggga taaataatat ttcatcttca t 31 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 31
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<210> SEQ ID NO 6 <211> LENGTH: 21 <212> TYPE:
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SEQ ID NO 10 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
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<400> SEQUENCE: 10 cttcagattt tcaataaggt a 21 <200>
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sequence <220> FEATURE: <223> OTHER INFORMATION: PCR
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ccacaccgtg c 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 12 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR oligonucleotide primer
<400> SEQUENCE: 12 gttgccgctg cagctacatt t 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 13 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Peptide sequence <400> SEQUENCE: 13 Ser Asp Asn Ala Thr Arg
Pro Ala Phe Ser Glu Arg Leu Ser Gln Met 1 5 10 15 Thr Asn
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Peptide sequence <400> SEQUENCE: 14 Phe Ser Arg
Val Lys Lys Ser Val Glu Val Leu Lys Asn Asn Lys Ala 1 5 10 15 Pro
Tyr <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Peptide sequence <400> SEQUENCE: 15 Glu Gln Pro
Ala Asn Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu 1 5 10 15 Lys
Ser <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 16
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Residues 99-116 of Mature hIL-9 Receptor <400>
SEQUENCE: 16 Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu
Glu Ile Phe 1 5 10 15 Gln Lys <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 17 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer <400>
SEQUENCE: 17 gtaaaacgac ggccagt 17 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 18 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide
primer <400> SEQUENCE: 18 gtaaaacgac ggccagt 17 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Mus musculus
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Murine IL-9 Receptor Peptide Sequence
<400> SEQUENCE: 19 Gly Gly Gln Lys Ala Gly Ala Phe Thr Cys 1
5 10 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: Murine IL-9 Receptor
Peptide Sequence <400> SEQUENCE: 20 Leu Ser Asn Ser Ile Tyr
Arg Ile Asp Cys His Trp Ser Ala Pro Glu 1 5 10 15 Leu Gly Gln Glu
Ser Arg 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 21 <211> LENGTH: 21 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: Murine IL-9 Receptor
Peptide Sequence <400> SEQUENCE: 21 Cys Glu Ser Tyr Glu Asp
Lys Thr Glu Gly Glu Tyr Tyr Lys Ser His 1 5 10 15 Trp Ser Glu Trp
Ser 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
22 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Residues 8-14 of Mature hIL-9 Receptor
<400> SEQUENCE: 22 Thr Cys Leu Thr Asn Asn Ile 1 5
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 23
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Residues 50-67 of Mature hIL-9 Receptor <400>
SEQUENCE: 23 Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr
Met Gln Thr 1 5 10 15 Arg Tyr <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 24 <211> LENGTH: 415 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24
tctcgagcag gggtgtccaa ccttggcggg gatcctggac atcaacttcc tcatcaacaa
60 gatgcaggaa gatccagctt ccaagtgcca ctgcagtgct aatgtgacca
gttgtctctg 120 tttgggcatt ccctctgaca actgcaccag accatgcttc
agtgagagac tgtctcagat 180 gaccaatacc accatgcaaa caagataccc
actgattttc agtcgggtga aaaaatcagt 240 tgaagtacta aagaacaaca
agtgtccata tttttcctgt gaacagccat gcaaccaaac 300 cacggcaggc
aacgcgctga catttctgaa gagtcttctg gaaattttcc agaaagaaaa 360
gatgagaggg atgagaggca agatatgaag atgaaatatt atttatccca gctgc 415
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 25
<211> LENGTH: 127 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 25 Gln Gly Cys Pro Thr Leu Ala
Gly Ile Leu Asp Ile Asn Phe Leu Ile
1 5 10 15 Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser
Ala Asn 20 25 30 Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp
Asn Cys Thr Arg 35 40 45 Pro Cys Phe Ser Glu Arg Leu Ser Gln Met
Thr Asn Thr Thr Met Gln 50 55 60 Thr Arg Tyr Pro Leu Ile Phe Ser
Arg Val Lys Lys Ser Val Glu Val 65 70 75 80 Leu Lys Asn Asn Lys Cys
Pro Tyr Phe Phe Ser Cys Glu Gln Pro Cys 85 90 95 Asn Gln Thr Thr
Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu 100 105 110 Glu Ile
Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile 115 120 125
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 26
<211> LENGTH: 415 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 26 tctcgagcag gggtgtccaa
ccttggcggg gatcctggac atcaacttcc tcatcaacaa 60 gatgcaggaa
gatccagctt ccaagtgcca ctgcagtgct aatgtgacca gttgtctctg 120
tttgggcatt ccctctgaca actgcaccag accatgcttc agtgagagac tgtctcagat
180 gaccaatacc accatgcaaa caagataccc actgattttc agtcgggtga
aaaaatcagt 240 tgaagtacta aagaacaaca agtgtccata tttttcctgt
gaacagccat gcaaccaaac 300 catggcaggc aacgcgctga catttctgaa
gagtcttctg gaaattttcc agaaagaaaa 360 gatgagaggg atgagaggca
agatatgaag atgaaatatt atttatccca gctgc 415 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 27 <211> LENGTH: 126
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 27 Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu
Asp Ile Asn Phe Leu Ile 1 5 10 15 Asn Lys Met Gln Glu Asp Pro Ala
Ser Lys Cys His Cys Ser Ala Asn 20 25 30 Val Thr Ser Cys Leu Cys
Leu Gly Ile Pro Ser Asp Asn Cys Thr Arg 35 40 45 Pro Cys Phe Ser
Glu Arg Leu Ser Gln Met Thr Asn Thr Thr Met Gln 50 55 60 Thr Arg
Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val Glu Val 65 70 75 80
Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn 85
90 95 Gln Thr Met Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu
Glu 100 105 110 Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys
Ile 115 120 125 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 28 <211> LENGTH: 585 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 28
atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgttgcgcaa
60 gctgactaca aggacgacga tgacaagctt gaattctcta gagatatcgt
cgacagatct 120 ctcgagcagg ggtgtccaac cttggcgggg atcctggaca
tcaacttcct catcaacaag 180 atgcaggaag atccagcttc caagtgccac
tgcagtgcta atgtgaccag ttgtctctgt 240 ttgggcattc cctctgacaa
ctgcaccaga ccatgcttca gtgagagact gtctcagatg 300 accaatacca
ccatgcaaac aagataccca ctgattttca gtcgggtgaa aaaatcagtt 360
gaagtactaa agaacaacaa gtgtccatat ttttcctgtg aacagccatg caaccaaacc
420 acggcaggca acgcgctgac atttctgaag agtcttctgg aaattttcca
gaaagaaaag 480 atgagaggga tgagaggcaa gatatgaaga tgaaatatta
tttatcccag ctgccaacgg 540 tagcgaaacc agccagtgcc actgcaatcg
cgatagctgt ctttt 585 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 29 <211> LENGTH: 168 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 29 Met
Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10
15 Thr Val Ala Gln Ala Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu Phe
20 25 30 Ser Arg Asp Ile Val Asp Arg Ser Leu Glu Gln Gly Cys Pro
Thr Leu 35 40 45 Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile Asn Lys
Met Gln Glu Asp 50 55 60 Pro Ala Ser Lys Cys His Cys Ser Ala Asn
Val Thr Ser Cys Leu Cys 65 70 75 80 Leu Gly Ile Pro Ser Asp Asn Cys
Thr Arg Pro Cys Phe Ser Glu Arg 85 90 95 Leu Ser Gln Met Thr Asn
Thr Thr Met Gln Thr Arg Tyr Pro Leu Ile 100 105 110 Phe Ser Arg Val
Lys Lys Ser Val Glu Val Leu Lys Asn Asn Lys Cys 115 120 125 Pro Tyr
Phe Ser Cys Glu Gln Pro Cys Asn Gln Thr Thr Ala Gly Asn 130 135 140
Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu Ile Phe Gln Lys Glu Lys 145
150 155 160 Met Arg Gly Met Arg Gly Lys Ile 165 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 30 <211>
LENGTH: 585 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 30 atgaaaaaga cagctatcgc gattgcagtg
gcactggctg gtttcgctac cgttgcgcaa 60 gctgactaca aggacgacga
tgacaagctt gaattctcta gagatatcgt cgacagatct 120 ctcgagcagg
ggtgtccaac cttggcgggg atcctggaca tcaacttcct catcaacaag 180
atgcaggaag atccagcttc caagtgccac tgcagtgcta atgtgaccag ttgtctctgt
240 ttgggcattc cctctgacaa ctgcaccaga ccatgcttca gtgagagact
gtctcagatg 300 accaatacca ccatgcaaac aagataccca ctgattttca
gtcgggtgaa aaaatcagtt 360 gaagtactaa agaacaacaa gtgtccatat
ttttcctgtg aacagccatg caaccaaacc 420 atggcaggca acgcgctgac
atttctgaag agtcttctgg aaattttcca gaaagaaaag 480 atgagaggga
tgagaggcaa gatatgaaga tgaaatatta tttatcccag ctgccaacgg 540
tagcgaaacc agccagtgcc actgcaatcg cgatagctgt ctttt 585 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 31 <211>
LENGTH: 168 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 31 Met Lys Lys Thr Ala Ile Ala Ile
Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp
Tyr Lys Asp Asp Asp Asp Lys Leu Glu Phe 20 25 30 Ser Arg Asp Ile
Val Asp Arg Ser Leu Glu Gln Gly Cys Pro Thr Leu 35 40 45 Ala Gly
Ile Leu Asp Ile Asn Phe Leu Ile Asn Lys Met Gln Glu Asp 50 55 60
Pro Ala Ser Lys Cys His Cys Ser Ala Asn Val Thr Ser Cys Leu Cys 65
70 75 80 Leu Gly Ile Pro Ser Asp Asn Cys Thr Arg Pro Cys Phe Ser
Glu Arg 85 90 95 Leu Ser Gln Met Thr Asn Thr Thr Met Gln Thr Arg
Tyr Pro Leu Ile 100 105 110 Phe Ser Arg Val Lys Lys Ser Val Glu Val
Leu Lys Asn Asn Lys Cys 115 120 125 Pro Tyr Phe Ser Cys Glu Gln Pro
Cys Asn Gln Thr Met Ala Gly Asn 130 135 140 Ala Leu Thr Phe Leu Lys
Ser Leu Leu Glu Ile Phe Gln Lys Glu Lys 145 150 155 160 Met Arg Gly
Met Arg Gly Lys Ile 165 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 32 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR oligonucleotide primer
<400> SEQUENCE: 32 gctccagtcc gctgtcaa 18 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 33 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: PCR
oligonucleotide primer <400> SEQUENCE: 33 ctccccctgc agcctacc
18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 34
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: PCR oligonucleotide primer <400> SEQUENCE: 34
cggggctgac taaaggttct 20 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR oligonucleotide primer
<400> SEQUENCE: 35 gttcttaaag agcattcact 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 36 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: PCR
oligonucleotide primer <400> SEQUENCE: 36 attttcacat
ctggaatctt cact 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR oligonucleotide primer
<400> SEQUENCE: 37 aatccaaggt caacattatg 20 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 38 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: PCR
oligonucleotide primer <400> SEQUENCE: 38 tttctttgaa
taaatcctta c 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 39 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR oligonucleotide primer
<400> SEQUENCE: 39 gaaatcacca acaggaacat a 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 40 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: PCR
oligonucleotide primer <400> SEQUENCE: 40 atcaactttc
atccccacag t 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 41 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR oligonucleotide primer
<400> SEQUENCE: 41 ggataaataa tatttcatct tcat 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 42 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Oligonucleotide primer <400> SEQUENCE: 42 caaatctgaa
gagcaaacta t 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 43 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer <400>
SEQUENCE: 43 ttaaaaaatt catttcagta ttct 24 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 44 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Kinase
end-labeled p32 oligonucleotide probe <400> SEQUENCE: 44
aattacctta ttgaaaatct gaag 24
* * * * *